620
ENZYMES
[27]
and effective strategy in synthetic organic chemistry. New transformations based on known or new enzymes will continue to be exploited. It is worth noting that the number of enzymes reported so far (about 2300) represents only approximately 2% of the total number of enzymes existing in nature. Many unknown enzymes remain to be explored. Known enzymes or proteins can be altered through site-directed mutagenesis or chemical or biological modification to provide new catalytic activities. New protein catalysts specific for a predetermined reaction are now available through the catalytic antibody approach. Multiple enzyme systems required for efficient synthesis of complex molecules such as antibiotics may be constructed within a cell via genetic manipulation and metabolic pathway engineering. It seems fair to say that virtually all kinds of protein catalysts can be constructed from the 20 common amino acids. Figure 14 illustrates our strategy for the development of a specific and efficient protein catalyst for the transformation of a desired reaction. Acknowledgments This work was supported by the National Institutes of Health (GM44154). We thank Professor Richard Lerner for advice on the work involvingFab libraries.
[2 7] M o d i f i c a t i o n o f E n z y m e C a t a l y s i s b y E n g i n e e r i n g Surface Charge
By GREGORIO ALVARO and ALAN J. RUSSELL Introduction The rational modification of enzyme catalysis has been an elusive goal of biochemists for many years.l Until the combination of the techniques of recombinant DNA methodologies and DNA sequencing enabled "protein engineers" to introduce specific changes in the amino acid sequence of proteins via site-directed mutagenesis,2 the alteration of enzyme characteristics was limited to chemical modification? Chemical alterations were rarely specific and often resulted in significant changes in protein structure. Ulmer's insightful review I in 1983 described the properties of enzymes which could be altered in order to "improve" biocatalyst properties, and l K . M. U l m e r , Science 219, 666 (1983).
2 M. J. Zollerand M. Smith,D N A 3, 479 (1984). 3A. Gounaris and M. Ottensen, C. R. Tray. Lab. Carlsberg 35, 37 (1965). METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
621
almost all these goals have now been reached. Site-directed mutagenesis is now a relatively straightforward procedure and is a common step in the analysis of cloned genes. The term "protein engineering" is also generally accepted to refer to the systematic replacement of amino acid residues in order to alter the properties of a protein. The importance of electrostatic interactions in proteins has been recognized for many years and has been excellently summarized by Warshel et al.4: " T o express the structures of proteins with their functions one must first be able to express structures in terms of energies. Probably the most important requirement for such a correlation is the ability to evaluate the energies of charges in proteins." The key role of surface charge in fine tuning the activity, specificity, stability, and pH dependence of enzymes has been the subject of extensive research since the early 1920s. LinderstrCm-Lang 5 and Kirkwood 6 and colleagues performed pioneering research in developing our understanding of the effect of surface charge on surrounding ionizable amino acid residues. There are currently two approaches to determining quantitatively the importance of charge-charge interactions in proteins: experimental and theoretical. This chapter is concerned with an experimental, rational method for the determination and manipulation of electrostatic effects in enzymes. It is important, however, to realize the limitations of both theory and experimentation in this field. Experimental predictability will result from an accurate theory describing electrostatic interactions, whereas an accurate theory will depend on the existence of experimental systems to test current models. Thus, the most information will be derived from analyses of extensively characterized systems, for which a structure has been determined, and from which the effect of a charge alteration can be measured without interference from conformational changes or altered ionizations. Since the activity and specificity of proteins is so dependent on electrostatic interactions, it is not surprising that protein redesign strategies have been directed at manipulating these types of interactions. Alteration of Surface Charge rather than Internal Charge The distribution of charged groups in proteins is such that over 95% of the charged groups are on the surface (accessible to solvent) and only 5% buried. 7 Furthermore, internal buried charges are usually critically 4 A. Warshel, F. Sussman, and G. King, Biochemistry 25, 8368 (1986). 5 K. LinderstrCm-Lang, C. R. Tray. Lab. Carlsberg 15, 70 (1924). 6 j. G. Kirkwood and F. H. Westheimer, J. Chem. Phys. 6, 506 (1938). 7 D. J. Barlow and J. M. Thornton, Biopolymers 25, 1717 (1986).
622
ENZYMES
[27]
important either catalytically, structurally, or both. For this reason any alteration of internal charge via mutagenesis would have unpredictable results. Surface charge, however, is readily amenable to chemical and site-specific mutagenesis. The first experimental tests of theoretical models for electrostatic interactions utilized chemical modification of charged surface residues to neutralize or reverse the charge on the surface of a variety of enzymes. 3'8-~4 Although chemical modification had significant effects on enzyme function, it was found to be an unpredictable method: similar changes in net surface charge in different enzymes had very different effects on activity and pH dependence) Even so, in 1980 Rees 15 investigated the effect of surface charge on the redox potential of cytochrome c, demonstrating that surface charge could be responsible for a significant portion of the redox control of such proteins. These results were criticized because Rees altered surface charge using chemical modification,~6 resulting in possible conformational changes or artifactual results. What remained clear was that alterations in surface charge resulted in somewhat predictable changes in the redox potential of the protein. Since, when designing an experiment to modify catalysis, it is important to be able to predict the approximate magnitude of any effect resulting from a change in protein structure, chemical modification of surface charge is not the method of choice. The more refined tool of site-directed mutagenesis enables the introduction or removal of charge at a defined point in a protein, allowing ultimate control over which point charges should be altered. Thus, the question now becomes what charge should be altered, and how much of a change in activity would be expected from such an alteration. L o n g R a n g e versus S h o r t R a n g e
There are basically two types of electrostatic interactions within proteins: long range (>5/~) and short range ( Ser-99 would minimize any large conformational changes, whereas the net surface charge would be altered. If data regarding sequence similarities are not available, there are still some useful guidelines which will minimize unexpected and uninterpret-
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
627
able results. First, the side chain which is inserted should be smaller than the one which is being removed. In general, it is far better to leave a hole in the protein after mutation rather than introduce large side chains which require tertiary structural changes in order to accommodate the new amino acid. Although changes on the surface of an enzyme will be less invasive than in the interior, it is still advisable to exercise caution in choosing the mutation to make. Second, if possible it is advisable to use structurally similar amino acids when designing mutations; such alterations are often described as "conservative." For instance, replacing glutamate (or even histidine) with glutamine would be unlikely to result in large conformational changes. Finally, the introduction of amino acids which could distort the backbone of the protein should also be avoided (in particular, proline and glycine residues). Selection o f Mutagenesis Method The general strategy of site-directed mutagenesis is shown in Fig. 1 and has been described previously. 2 The synthetic mutagenic oligonucleotide (previously phosphorylated) is annealed to the viral M13 template DNA containing the gene to be mutated. A DNA polymerase (Klenow fragment, lacking the proofreading function) is then used in in vitro complementary strand synthesis, and DNA ligase is used to ligate the nascent chain to the end of the mutagenic oligonucleotide, forming a partial heteroduplex. This DNA is then used to transfect competent Escherichia coli, giving rise to plaques with either the mutant or wild-type gene. The mutant plaques must then be identified by either colony-blot hybridization, z5 direct plaque screening, or sequencing. After selection of mutant clones, the gene must be fully sequenced to ensure that only the desired mutation has been introduced. A serious but common error in mutagenesis is to sequence the region of the mutation, rather than the entire gene. The efficiency of mutagenesis is affected by a variety of factors. One of the most important of these is the ability ofE. coli to repair the mismatch which has been introduced during mutagenesis. If the two strands could not be distinguished by the host cell then there is a 50% chance that the repair process will correct in favor of the mutant strand. However, M13 DNA synthesized in vivo will be methylated by the enzyme deoxyadenosine methylase, which methylates adenosines in a GATC sequence. Mismatch repair enzymes use this methylase activity in proofreading, 26 and after DNA replication in vitro the newly synthesized strand will be under25 R. B. Wallace, M. J. Johnson, T. Hurose, T. Miyaka, E. H. Kawashima, and K. Itakura, Nucleic Acids Res. 9, 879 (1981). 26 A. Lein-Lu, S. Clark, and P. Modrich, Proc. Natl. Acad. Sci. U.S.A. 82, 7840 (1983).
628
ENZYMES
~
[27]
Mutagenic Oligonucleotide
L
DNA Polymerase(Klenow) dNTP's DNA Ligase
I Transform Screen Select
FIG. 1. General strategy for site-directed text.
mutagenesis. F o r
a detailed explanation, see
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
629
methylated. Thus, the host cell can distinguish between the parent and daughter strand in favor of the parent. To increase the efficiency of mutagenesis (which is necessary to replace hybridization screening with sequencing), many of the strains used in mutagenesis strategies are deficient in mismatch repair functions (mutL mutants27). Experimental Procedures
Site-Directed Mutagenesis There are now many site-directed mutagenesis techniques which are relatively straightforward. Indeed, once a gene has been cloned into the bacteriophage MI3, it is possible to purchase kits which guarantee mutagenesis efficiencies in excess of 50%. Rather than presenting all the alternatives (these have been described in detail elsewhere28), we discuss the most important factors governing mutagenesis yields, and a typical strategy is outlined. The primary factor governing the success of a mutagenesis experiment is the quality and choice of oligonucleotide. In particular, low-purity oligonucleotides or otigonucleotides which do not bind specifically to the template DNA can result in extremely low yields of mutants. Care must be taken when designing oligonucleotides to ensure absolute specificity of binding. It is advisable to check the oligonucleotide in a sequencing reaction for sequencing capability prior to use. This gives a good indication as to whether the oligonucleotide is pure and specific enough to be used. Once pure template has been obtained as described previously,27 the mutagenesis reaction sequence is initiated with the annealing of oligonucleotide and template. The purity of template is as important as that of the oligonucleotide. Annealing. One hundred picomoles of primer (oligonucleotide) is kinased by incubating the following mixture for 30 min at 37° and then 10 min at 70 °. 10/zl Primer 2 /zl 10 × KB buffer [500 mM Tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.5, 100 mM magnesium chloride, 50 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM spermidine hydrochloride] 1/zl Dithiothreitol (100 mM) 27 p. Carter, H. Bedouelle, M. M. Y. Waye, and G. Winter, "Oligonucleotide Site-Directed Mutagenesis in M I 3 . " Anglian Biotechnology Limited, 1985. 28 R. J. Leatherbarrow and A. R. Fersht, Protein Eng. 1, 7 (1986).
630
ENZYMES
[27]
2/zl Adenosine triphosphate (10 mM) 4/zl Sterile water 1/xl T4 polynucleotide kinase (0.5 units//xl) One microgram of M13 template DNA should then be added to 10 pmol of kinased primer in a final volume of 10 t-d (1 x TM: 100 mM Tris-HC1, pH 8.0, 100 mM magnesium chloride), placed in a beaker of hot water (80°), and allowed to cool to room temperature ( - 2 0 min). Extension and Ligation. To the 10/zl of annealing mix, the following should be added: 1/xl 10 x TM buffer 1 /xl Deoxynucleotide triphosphates (5 mM each of adenosine, guanine, cytosine, and thymine) 1/zl Dithiothreitol (I00 mM) 4/~1 Sterile water 1/xl T4 DNA ligase (10 units//zl) 0.5/xl DNA polymerase, Klenow fragment (5 units//xl) Incubation is conducted overnight at 15° before the mixture is used to transfect E. coil as described previously. 29 Plaque Screening. Plates containing approximately 250 plaques can then be used in direct plaque screening, if the transformations have been performed using H-Top agarose instead of H-Top agar. Place a circular nitrocellulose filter disk on the plate; remove the disk after 5 min, and bake the filter for 2 hr. The filter should then be prewetted in 6 x SSC buffer [from a 20 x SSC stock (17.5% (w/v) sodium chloride, 8.8% (w/v) sodium citrate, pH 7.0)] for 5 min in a sealed plastic bag and prehybridized at 67 ° for 2 hr. After rinsing the filters in 6 x SSC, they should be placed in another plastic bag with 10 pmol radiolabeled mutagenic oligonucleotide, incubated at 67 ° for 30 min, and allowed to cool to room temperature overnight. The filters should then be washed in buffer, dried, and autoradiographed. Starting at 5° below the melting temperature for the oligonucleotide (as calculated from Wallace's rules29), this cycle of washing and autoradiography should be performed at increasing temperatures (with 2° increments) until mutant and wild-type colonies can be distinguished. Once mutant plaques have been identified, they can be used in preparation of template DNA for sequencing 3° and eventually subcloning into an ex29 S. V. Suggs, T. Hirose, T. Miyake, E. H. Kawashima, M. J. Johnson, K. Itakura, and R. B. Wallace, in "Developmental Biology Using Purified Genes" (D. Brown, ed.). Academic Press, New York, 1981. 30 A. T. Bankier and B. G. Barrell, in "Techniques in the Life Sciences B5, Nucleic Acids Biochemistry, B508." Elsevier, Ireland, 1983.
[2 7]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
631
pression vector. Expression and purification of the protein will enable characterization of the effects of the electrostatic alterations. Techniques such as casette mutagenesis 31 and the polymerase chain reaction 32 have not been discussed here, but they will undoubtedly impact greatly on the ease of mutant protein screening and production. The availability of mutagenesis kits has led to a dramatic reduction in the time necessary to make mutants. However, the analysis of each mutant is the critical part of determining structure-function relationships in proteins, and the temptation to produce large numbers of mutants, without a detailed investigation of the properties of each, must be resisted. Importance of Plaque Purification and Sequencing. Once a plate containing many plaques has been generated, there is a temptation to perform template DNA preparations for sequencing immediately. This will often lead to a mixed population of template DNA, since each plaque can contain both wild-type and mutant DNA (as a result of bacteriophage migration in the plate). Another unfortunately common practice is to sequence only the region of DNA surrounding the site of mutation. It is absolutely necessary to sequence the entire coding region of the protein if interpretation of the data is to be valid.
Characterization of Mutant Proteins In order to present a meaningful explanation for the properties of mutant proteins with altered surface charge, the wild-type and mutant proteins should be subjected to extensive kinetic (and, if possible, structural) investigations. Although the methods used in characterizing mutant proteins will be different for each family of enzymes (and are beyond the scope of this chapter), the following section provides comments which are important for the analysis, and interpretation, of all systems. Need for Active-Site Titration. Of utmost importance is the need for a pure sample of the mutant protein, free of contamination from wild-type or other mutant proteins. If a mutant protein has reduced activity relative to wild type, then the presence of small amounts of wild-type protein could result in a false interpretation of the activity of a mutant. The most simple way to determine if the activity present in a sample of mutant enzyme is the result of only a small fraction of the total protein is with an active-site titration. Much of the work in the area of protein engineering of surface charge has been performed with proteases, and quantitative knowledge of the concentration of all reacting species is particularly important because of the problem of autolysis. Titrating enzyme with a variety of small 31 j. A. Wells, M. Vasser, and D. B. Powers, Gene 34, 315 (1987). 32 H. A. Ehrlich, " P C R Technology." Stockton Press, New York, 1989.
632
ENZYMES
[27]
organic molecules avoids the internal inconsistency of using a rate assay to calibrate a method which uses a rate assay as its absolute standard. The organic active-site titrants have the property that their reaction with the enzyme is dependent on the integrity of the active center. Thus, the titration depends not on the enzyme itself, but rather on the substrate. For instance, the protease subtilisin can be titrated with N-trans-cinnamoylimidazole as described in detail previously. 33 Unfortunately, activesite titrations are not available for most enzymes, although an assay could be developed if a tight-binding competitive inhibitor can be identified. In such cases, reacting the enzyme with radiolabeled inhibitor and recovering the complex would enable the calculation of the concentration of enzyme from the amount of radiolabel present after the removal of unbound titrant. The active-site titration of tyrosyl-tRNA synthetase (tyrosine-tRNA ligase), the first enzyme to be subjected to site-directed mutagenesis, is performed in such a way and has been described previously. 34 Finally, it is important to point out that a careful kinetic investigation of mutant and wild-type enzymes will be meaningless in the absence of good data analysis techniques. In particular, the use of linear regression to solve Lineweaver-Burke plots (which enable the "determination" of kcat and Km is totally unacceptable. There are now a number of excellent computer programs (which are as user-friendly as a calculator) that perform nonlinear regression on the original data. 35 Nonlinear regression of data does not distort the experimental error in kinetic determinations and should be used in all instances. In general, computational analysis of data is faster and more accurate than fitting by eye (which is still performed in some laboratories), but it is always necessary to understand the limitations of the computer and to remember that a computer will fit even the worst data to a given equation. Dielectric Constant and Effect of Ionic Strength. This chapter suggests that by altering surface charge it is possible to manipulate biocatalytic behavior in a rational manner. Such a method depends on the strength of electrostatic interactions between the surface of a protein and the amino acids that are directly involved in binding and catalysis, which in molecular terms are far from the site of mutation. The strength of an electrostatic
33 M. L. Bender, M. L. Begoue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kezdy, T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966). 34 A. R. Fersht, J. P. Shi, J. Knill-Jones, D. M. Lowe, A. J. Wilkinson, D. M. Blow, P. Brick, P. Carter, M. M. Y. Waye, and G. Winter, Nature (London) 314, 235 (1985). 35 " E N Z F I T T E R " and " G R A F I T " were written by R. J. Leatherbarrow (Imperial College, London), and are specifically designed for enzyme data.
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
633
interaction will depend on the ability of the material separating the charges to attenuate the interaction. For homogeneous materials the ability of a material to polarize an electric field is described by the dielectric constant (D). Free space, by definition, has a dielectric of I, while pure water has a dielectric of 78.5 at 25 °. D is a macroscopic quantity which varies inversely with temperature. When small diffusible ions are included in a solvent, the dielectric for the solution increases and is inversely related to the separation distance between two point charges in the medium. Hill 36 defined this Debye-Htickel screening 37 as an effective dielectric constant (Deer), which is related to the dielectric of water (Dw) as follows:
Deer = D w ( e kr) where k is the Debye-Hiickel parameter, which is proportional to ionic strength, and r is the separation distance between the point charges. Although the applicability of the concept of dielectric constants to proteins is discussed later, there is no question that the strength of an electrostatic interaction will depend on the ionic strengh of the solvent. Thus, any modification of enzyme properties as a result of alteration of surface charge should also be dependent on ionic strength: increasing ionic strength should decrease the effect of the mutation. Testing the effect of ionic strength on the difference between mutant and wild-type proteins is necessary for protein engineering strategies which depend on the alteration of surface charge. Figure 2 shows the dependence of the difference between the PKa of a surface charge mutant of subtilisin and wild-type subtilisin on ionic strength. 38 Note that as ionic strength increases, the effect of the mutation becomes negligible. If such a test does not demonstrate a dependence on ionic strength, it is unlikely that the effect of the mutation is a result of an altered electrostatic interaction. From the preceding discussion it is obvious that the effect of a change in surface charge will be maximized at low ionic strengths, and experiments should be designed with this in mind. Experimental Determination of Dielectric Constants in Proteins. As pointed out above, the strength of electrostatic interactions in proteins will be a function of the dielectric constant in the region which separates the charges. Thus, in order to predict the effect of a change in surface charge on a distant charged amino acid, the dielectric constant must be determined. In the same way that the strength of hydrogen bonds 34 and 36 T. L. Hill, J. Phys. Chem. 60, 253 (1956). 37 p. Debye and E. Hiickel, Phys. Z. 24, 185 (1923). 38 A. J. Russell and A. R. Fersht, Nature (London) 328, 496 (1987).
634
ENZYMES
[27]
0.5
~"
0.4
03 I
0.3 4-) "o ,-.t -~
0.2
Q., 0.1
0.0 0.001
0.005
0.01
Ionic
0.025
Strength
0.1
0.5
1
(M)
FIG. 2. Dependence of ApKa of histidine-64 between wild-type and DS99 subtilisins on ionic strength. The Ap Ka values represent the difference in PKa values for the pH dependence of kcat/K m between wild-type and aspartate ~ serine-99 (DS99) subtilisins in the hydrolysis of succinyl-L-alanyl-L-alanyl-L-prolyl-t-phenylalanyl-p-nitroanilide. The assays were performed at the ionic strengths indicated, as described previously [A. J. Russell, P. G. Thomas, and A. R. Fersht, J. Mol. Biol. 193, 803 (1987); P. G. Thomas, A. J. Russell, and A. R. Fersht, Nature (London) 318, 375 (1985)]. Note that the 0.001 M experiments utilized imidazole buffers, whereas the remaining experiments were performed with phosphate buffers.
hydrophobic interactions 39 have been measured by their removal through mutagenesis, the energy of an electrostatic interaction can also be calculated from the effect of its removal. As for any determination of energies from differences between mutants, these free energies are apparent free energies representing the sum of one or more energy changes. Thus, the effect of a small perturbation in enzyme structure which removes (or implants) an electrostatic interaction will enable the calculation of the apparent free energy of that particular interaction. In addition, if the kinetic effect of the mutation can be localized to an effect on one particular charged residue, and the distance separating the surface point charge and 39 j. T. Kellis, K. Nyberg, D. Sali, and A. R. Fersht, Nature (London) 333, 784 (1988).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
635
that residue is known, then an apparent dielectric constanP ° for the region between these charges can be estimated. By systematically replacing and introducing charged surface residues, it should be possible to map the dielectric constant through different regions of the protein. The temptation is to use one value of dielectric for the whole protein, since this simplifies the system considerably, but this type of uniform continuum electrostatic model is obviously a gross oversimplification of reality. Also, in terms of an experimental determination of dielectric for a particular interaction, it is crucial to remember that this value will refer only to that specific interaction and should not be used to describe the whole protein. In referring to the dielectric properties of proteins Rogers 41 has likened proteins to Swiss cheese, that is, there appear to be lines of hydrophilicity (and hence high dielectric) within the generally hydrophobic interior of globular proteins, demonstrating the ease with which data can be overinterpreted. In addition, as Warshel and Russell 42 have pointed out, charged groups do not only interact with each other, but also with the protein dipoles. Calculations using experimental data should take into account these interactions wherever possible. With all these restrictions and caveats in mind for the interpretation of experimental data, there may seem little point in calculating a value of the dielectric. However, without knowledge of the screening of charges within a protein and at the protein-solvent interface, understanding of the electrostatic properties of proteins cannot develop. The term apparent dielectric constant has been proposed 4° to describe dielectric constants calculated from mutant protein data. This term stresses the local nature of dielectrics within a protein, while at the same time retaining the concept of a dielectric constant. Control Mutations. The mere confirmation that an enzyme has altered properties as a result of a change in surface charge does not conclusively prove that the effect is a result of a modified electrostatic interaction. In order to avoid misinterpretation of data, it is advisable to repeat a surface charge alteration at a site distant from that of the first mutant. In addition, "control" mutations should be generated at each site. For instance, if an aspartate to serine mutation is introduced into the subtilisin sequence at position 99 (see Table I), a suitable control mutation would be the introduction of a lysine at the same position (thus doubling the change in net surface charge). The need for control mutations is particularly ira-
4o A. J. Russell, P. G. Thomas, and A. R. Fersht, J. Mol. Biol. 193, 803 (1987). 41 N. K. Rogers, Prog. Biophys. Mol. Biol. 48, 37 (1986). 42 A. Warshel and S. T. Russell, Q. Rev. Biophys. 17, 283 (1984).
636
ENZYMES
[27]
portant given that the strategy outlined herein will generate small, but measurable, changes in enzyme function. Ideally, the structure of each mutant should be determined in order to show that alterations in enzyme properties are not due to fortuitous changes in protein structure. Where this is not possible, further mutations and detailed kinetic analysis will aid interpretation of the results. For instance, mutation of charged surface residues could damage metal ion binding sites (in proteases these are particularly important for stabilizing the enzyme). Kinetic analysis of the stability of mutants in the presence and absence of such metal ions will indicate whether a change in activity is an indirect effect of an alteration in metal binding.
A p p l i c a t i o n s of S u r f a c e C h a r g e M o d i f i c a t i o n for A l t e r i n g Enzyme Properties
pH Dependence, Specificity, Stability, and Activity The first use of site-directed mutagenesis to tailor the properties of enzymes by modification of surface charge was directed at altering the pH dependence of subtilisin. 43 Mutant subtilisins were generated with shifted pH-activity profiles, higher catalytic activities, and altered specificities. Subtilisin is a serine protease which can catalyze the hydrolysis of amide or ester bonds via the acyl-enzyme mechanism. Because subtilisin is so well characterized both structurally and mechanistically, it has been described as an enzyme "designed to be engineered."44 During catalysis the imidazole side chain of His-64 acts as a general base. Thus, subtilisin is only active when His-64 is in the unprotonated state, and the activity of the enzyme follows exactly the pKa of this residue; in other words, the pK a of the enzyme (as can be measured via the pH dependence ofkcat/Km) is equivalent to the macroscopic pKa of His-64. Since the protonated form of His-64 will be stabilized by negative charges (even those distant from the active site), the neutralization of such charge should increase activity at low pH, and thus decrease the PKa of the enzyme. This hypothesis was tested using site-directed mutagenesis. The mutations introduced were at surface residues 99 and 156 (both of which have negatively charged side chains in the wild-type enzyme) of subtilisin BPN'. In each case the amino acid was replaced by a serine (with a neutral side chain) and a lysine residue (with a positively charged side chain). The effect on the pH dependence of catalysis was studied by following the pH 43 p. G. Thomas, A. J. Russell, and A. R. Fersht, Nature (London) 318, 375 (1985). 44j. A. Wells and D. A. Estell, Trends Biochem. Sci. (Pers. Ed.) 13, 291 (1988).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
637
1.2_
1.0
?i!i;i!i!i!i!i!i:?!i~i~ ~i'i:i:i~:;i!i:i;iii;i': i~i:i:i~i:iii?i::?:: iiii:i~i~i:i~iii:i:::i'i ii;i;ii;iiiiiii?:~i;3~ !iiii?ii~;:ii;:.~ii;i iii:?:iiiiiii:;ii:i-iiii ::::::::::::::::::::: :::::::::::::::::::::::
0.8
?!i~i:i!ili:i!i~??:'i iiii?i:iii~i:i!i~i:i:
""
i!i!i!i!i!ili!i!i!i!i!i!!!i!!!
0.6
!i!i;i!i;i~i~-~iiii-iiii !i!iii:iiiiii:iii-?iiii
::::::::::::::::::::::::::
??????i~?i????:i
0.4
ii!iiiiiiii:i-iii~L:i
ii:ilii~ili;:i;i;:i:ili
:::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::::
!i~i!i!i:i!i!i~i~ili~!~;ii~ii!
::::::::::::::::::::::::::::
i!i!iii:i~i!i!i!i~i!i i~!ii~ii ::::::::::::::::::::::::::::
i:i:ii?:i:: :::: i3:i:?i:?:: ?ii: ::i:i: i ?i~i:i ?i
i~iiiii?i~iiii!ili!i!i~ii:~ii iiiiiiiiiiiiii
::::::::::::::::::::::::::::::
::::::::::::::::::::::::::::
0.2
iiiiiiiiiiii!i~i!iiiiiii)iiii; iiiiiiii!iiiiiiiiiiiiiiili!iii
:::::::::::::::::::::
::::::::::::::::::::::::::::::: :i:i:iii!iiiii!i!!~iii!!iiiiii :+:+:+x+>::+:+ x+:+x:+:+>:+: :::::::::::::::::::::::::::::: :i:i:?:i:i:i:i•i-i-i:i•3!:i:i :::::::::::::::::::::::::::::
:::::::::::::::::::::::::::::::::::::::::
0.0
|
D-$99
|
E-S156
|
i
E-S,D-S
D-K99
E-K156
ENZYME FIG. 3. Shift in the pK a of histidine-64 of mutant subtilisins, relative to wild type, at 0.001 M ionic strength. The APKa values given are for the pH dependence of keat[K m in the hydrolysis of succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-p-nitroanilide. The assays were performed at 25 ° in imidazole-imidazolium buffer (ionic strength 0.001 M). The naming of the enzymes is such that the first letter represents the residue which has been mutated to a different amino acid (given by the second letter) at a particular position (given by the number).
dependence of kcat/Km; this measures the apparent PKa of the active-site histidine (His-64). In contrast to earlier studies using chemical modification to alter pH dependence which showed relatively small shifts, removal of just one surface carboxylate in subtilisin (Asp-99) lowers the pKa of its active-site histidine by 0.3 units, despite the groups being approximately 1.3 nm apart. Similar results were obtained on removal of a negative charge at Glu-156, which is approximately 1.4 nm away from His-64. These shifts in pKa are cumulative on sequential or simultaneous changes in charge. Double mutants show increased effects, as do single mutants which have a double charge change. Indeed, in the case of a quadruple charge change, the PKa of activity is decreased by 1 pH unit (see Fig. 3). The shifts in pK~ are
638
ENZYMES
[27]
ionic strength dependent (as shown in Fig. 2), as would be predicted for long-range electrostatic interactions. The detailed kinetic investigation of these mutant enzymes has also given insight on dielectric properties at the protein surface, the role of ions in electrostatic shielding, and field effects on catalysis. From the changes in pH dependence of catalysis it has been possible to determine the magnitude of an electrostatic interaction between two point charges, as well as to determine the effect of these mutations on specificity and activity. In addition, counterion binding to charged groups was shown to be important when interpreting kinetic data. The shifts in pK a of the mutant enzymes enable the calculation of the apparent free energy of interaction between His-64 and the mutated residue (AAG). It is particularly important to point out that this energy is calculated from the removal of an interaction, and thus is influenced by other factors. This is analogous to the calculation of the apparent free energy for a hydrogen bond using protein engineering. 34 These values for AAG are in the region of 0.5 kcal/mol for the interaction of two charges separated by approximately 1-1.5 nm. The analogous value for a hydrogen bond, over a distance of 0.3 nm, is approximately 1 kcal/mol (depending on the type of hydrogen bond). These values demonstrate the great importance of electrostatic interactions in proteins. From determinations of AAG it was possible to calculate the apparent dielectric constant for the region separating the two chargesY These calculations are subject to even more assumptions than those involved in estimating the original interaction energy. Although the calculations are an oversimplification, the results indicate that the dielectric constant in the region of the active-site cleft of subtilisin is between 40 and 50, approaching that of water. Furthermore, the relationship between measured dielectric and ionic strength fits the relatively simple Hill model 36 for electrostatic interactions. This does not mean that the Hill assumptions are correct, but rather that the data can fit the approximation, lending credence to the high value of dielectric observed. Finally, it is important to reemphasize the warning of Hill, namely, that the effective dielectric constant between any two points in a system such as a protein is a unique value for those two points and is not applicable to any other pair of points. Although these mutants were not designed to alter specificity and activity, a number of interesting correlations were observed. Glu-156 is located close to the substrate binding site of subtilisin, and as would be predicted the removal or reversal of negative charge at this position
45 N. Bjerrum, Z. Phys. Chem. Stoechiom. Verwandtschaftsl. 106, 219 (1923).
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increases the affinity of the enzyme for negatively charged substrates while decreasing the affinity for positively charged ones. Indeed, this was the first demonstration of the control of substrate specificity by alteration of distant surface charge. The study of subtilisin has shown that, as predicted by the classic LinderstrCm-Lang theory, 5 making the surface charge of a protein more negatively charged raises the pK a of acidic groups (they lose a positively charged proton on ionization). Conversely, making the surface more positively charged lowers the pKa of surrounding acidic groups. Since these changes are due to electrostatic effects, they will be maximized at low ionic strengths. Significant effects will, however, be manifested at ionic strengths as high as 0.1 M if multiply charged counterions are avoided. Indeed, mutations should be designed such that they do not concentrate counterions in the active-site cavity. It would be foolish to assume that all systems will be as predictable in their response to surface mutations as subtilisin has been. This work has, however, shown again the importance of long-range electrostatic interactions, and how via their manipulation the properties of a protein can be significantly and predictably altered. The pH-activity dependence of lysozyme has also been predictably altered by engineering of surface charge .46 In this instance the enzyme was redesigned to be a more efficient catalyst than wild type under conditions of high pH and ionic strength (the mutation used introduced positive charge at only two positions on the surface). In addition, the removal of positive charge at two surface residues enabled the generation of a mutant enzyme which could function at low pH and ionic strength. Removal and introduction of surface charge has also been used to test structure-function relationships in an enzyme. Watson and colleagues 47 have shown that an arginine residue on the surface of phosphoglycerate kinase is important for catalysis, even though it is situated 1.3 nm from the active site. This has been used to infer that, during catalysis, a large conformational change takes place which will bring the arginine into close proximity with the active site. Mutation of surface residues has also led to important advances in the analysis of charge effects on protein-protein interactions. 48'49 Naturally,
46 M. Muraki, M. Morikawa, Y. Jigami, and H. Tanaka, Protein Eng. 2(1), 49 (1988). 47 p. A. Walker, J. A. Littlechild, L. Hall, and H. C. Watson, Eur. J. Biochem. 183, 49 (1989). 48 j. E. Long, B. Durham, M. Okamura, and F. Millet, Biochemistry 28, 6970 (1989). 49 p. C. Weber, T. J. Lukas, T. A. Craig, E. Wilson, M. M. King, A. P. Kwiatkowski, and D. M. Watterson, Proteins: Struct. Funct. Genet. 6, 70 (1989).
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interactions between proteins occur at the surface, and thus the role of charged amino acids will be significant. It is important to realize, however, that these interactions are generally short range. Charged surface residues which are located close to the substrate binding site of subtilisin have also been used to design substrate specificity in subtilisin, s° Long-range electrostatic interactions are also important when considering the stability of proteins. Indeed, it has been shown that such interactions influence the stability and cooperativity of dihydrofolate reductase.51 Finally, surface charge mutations have also been used to dissect the charge effects in protein-metal binding: 2 ion transport: 3 ion binding, 54 and isolated helical structures) 5 Solvent Resistance
The surface amino acid side chains of proteins are not only important in fine tuning the electrostastic nature of the active site (as demonstrated by the work described above), but they also perform the central role in protein-solvent interaction. Solvent structure is of such importance that in order to interpret mutagenesis results completely, alterations in the organization of bound water molecules must be considered. This is of particular interest when considering strategies designed to enable proteins to function in low water (the typical solvent for enzymes) environments. There are several factors that necessitate the use of organic solvents as media for enzyme-catalyzed reactions. 56 First, most hydrophobic reagents of interest to synthetic chemists are soluble only in organic solvents. Second, an organic bulk solvent can shift the thermodynamic equilibrium of many hydrolysis reactions (e.g., of esters and peptides) to synthetic ones. Third, the use of organic solvents obviates the danger of bacterial contamination, which is a major problem in many aqueous biocztalytic reactors. Because such an approach is attractive, the field of enzyme
50 j. A. Wells, D. B. Powers, R. R. Bott, T. P. Graycar, and D. A. Estell, Proc. Natl. Acad. Sci. U.S.A. 84, 1219 (1987). 51 K. M. Perry, J. J. Onuffer, M. S. Gittleman, L. Barmat, and C. R. Matthews, Biochemistry 28, 7961 (1989). 52 S. R. Martin, S. Linse, C. Johansson, P. M. Bayley, and S. Fors~n, Biochemistry 29, 4188 (1990). 53 R. MacKinnon, R. Latorre, and C. Miller, Biochemistry 28, 8092-8099 (1989). 54 S. Linse, P. Brodin, C. Johansson, E. Thulin, T. Grundstrom, and S. Fors6n, Nature (London) 335, 651 (1988). 55 D. Sali, M. Bycroft, and A. R. Fersht, Nature (London) 335, 740 (1988). 56 A. Zaks and A. J. Russell, J. Biotechnol. 8, 259 (1988).
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catalysis in organic solvents (first reported in the 1960s 57) has undergone rapid expansion, particularly since the mid 1980s. The use of enzymes in organic solvents is becoming the norm for applied enzymologists and even synthetic organic chemists, 58 and naturally there is much interest in designing proteins with improved properties for use in hydrophilic organic solvents. There is little question that some water is an absolute necessity for the maintenance of the catalytic integrity of enzymes. Indeed, enzyme structure and function rely on the direct and indirect role of water in all the noncovalent interactions. 59 Although the need for water is obvious, the amount of water required in enzymatic systems is less clear. The estimation of the thickness of the water layer around the enzyme is subject to speculation because the minimum amount of water necessary to allow enzymes to function has been determined for relatively few enzymes. 6° The hydration of proteins consists of a number of distinct stages. For lysozyme, water molecules first gather around charged groups and then cluster and grow over most of the protein surface. When only half of the protein surface is covered in water molecules the enzyme is partially active. Thus, the total amount of water necessary for lysozyme activity is relatively low as long as the water is associated with the enzyme surface. It is the concentration of water on the enzyme which determines specific activity, and it has been proposed that the major factor in determining activity in nonaqueous media stems from the ability of a particular solvent to strip water from the enzyme. 6~ Importantly, there is no question that the amount of water required for optimal activity in organic solvents is enzyme-specific. This phenomenon means that some enzymes are "better" than others in organic solvents in that they can function in very low water environments in hydrophilic media. For instance, subtilisin is the only enzyme known which can function in anhydrous dimethylformamide. It seems obvious that proteins with modified behavior in organic solvents will have altered surface charge, since it is the charged surface residues which interact most strongly with solvent. It has been suggested that the best method to achieve stability of a correctly folded protein in an anhydrous environment is with the use of a set of "design rules. ''62 57 F. R. Dastoli and S. Price, Arch. Biochem. Biophys. 122, 289 (1967). 58 j. B. West, W. J. Hennen, J. L. Lalonde, J. A. Bibbs, Z. Zhong, E. F. Meyer, and C.-H. Wong, J. Am. Chem. Soc. 112, 5313 (1990). 59 G. E. Schulz and R. H. Schimer, in "Principles of Protein Structure." Springer-Verlag, New York, 1979. 6o j. A. Rupley, E. Gratton, and G. Careri, Trends Biochem. Sci. (Pers. Ed.) 8, 18 (1983). 61 A. Zaks and A. M. Klibanov, J. Biol. Chem. 263, 3194 (1988). 62 F. H. Arnold, Protein Eng. 2, 21 (1988).
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Arnold's design rules predict that the removal of unpaired surface charges will stabilize proteins owing to the energy gained by removal of an unfavorable interaction between a charged group and a relatively low dielectric solvent. This is in agreement with all the electrostatic models mentioned above. There are two problems with this approach that may, however, hinder rapid progress (as has been recognized by Arnold62). If it becomes necessary to remove most of charged groups in order to stabilize the protein, there will be difficulty expressing the new hydrophobic protein. This may not be a significant problem if it turns out that only a few charge neutralizations are necessary to stabilize the protein significantly. The major foreseeable problem will be the effect of the mutations on the activity of the enzyme. As described above, electrostatic interactions (which actually encompass all interactions) throughout the protein will be significantly affected by changes in the dielectric environment of the protein. Thus, it would not be surprising if the removal of surface charge, which would result in a close interaction between organic solvent and protein, were to disrupt the finely tuned electrostatic interactions with control the catalytic mechanisms of most enzymes. Naturally, the experimentation required to test the deisgn rules is being performed by Arnold. 63 An alternative approach (currently being investigated in our laboratory) is to introduce more charged residues onto the surface of the enzyme while keeping the net surface charge constant. This should enable the enzyme to retain a "thicker" layer of water when placed in organic solvents. This layer of water could then act as a high dielectric shield, effectively protecting the protein molecule from bulk solvent. The success of this approach will depend on the relative partitioning of water between an enzyme and a given solvent, and whether a more charged enzyme will shift the partitioning of water in the solvent from that solvent to the enzyme surface. Concluding Remarks The huge potential of protein engineering is demonstrated by the massive proliferation in investigations utilizing this technology since the first experiments of the early 1980s. 64 The ability to alter specifically one or more residues in a protein at will is such a powerful tool that it enables the investigator to determine energies of interaction between amino acid side chains within a protein. This information can then be used in the ab 63 F. H. Arnold, personal communication (1990). 64 G. Winter, A. R. Fersht, A. J. Wilkinson, M. Zoller, and M. Smith, Nature (London) 299, 756 (1982).
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initio design of new proteins which could catalyze novel processes. Indeed, the information derived from protein engineering studies has already been utilized in the design of a chemically synthesized " e n z y m e " with chymotrypsin-like properties. 65 The most obvious prerequisite for site-directed mutagenesis is that the enzyme under investigation has been cloned, sequenced, and expressed in a suitable system. The technique is, however, at its most powerful when a detailed knowledge of structure and function is available. Only in the light of such information will mutagenesis experiments yield information on the crucial relationships between structure and function. In many cases it is a relatively simple step to clone and express a given protein, and thus the rate-limiting step for protein engineers is in the analysis of the mutants generated. Acknowledgments Much of the work described in this chapter was performed by Dr. P. G. Thomas in the laboratory of Professor A. R. Fersht, and this review would not have been possible without their contributions. The work was funded by Science and Engineering Research Council, UK. We also thank Dr. F. H. Arnold for helpful discussions about designing solvent-resistant enzymes. The solvent-resistant enzyme project of A.J.R. and G.A. is currently funded by grants from the National Science Foundation (BCS-9057312), the Petroleum Research Fund (Type G: 22019-G4), and the University of Pittsburgh Internal Grant Program. G.A. is a Spanish Ministry of Science and Education Postdoctoral Fellow. 65 K. W. Hahn, W. A. Klis, and J. M. Stewart, Science 248, 1544 (1990).
[28] M u t a t i o n a l R e m o d e l i n g o f E n z y m e S p e c i f i c i t y
By ROGER BONE and DAVID A. AGARD Introduction Limiting the range of substrates which are catalytically productive is one of the fundamental functions that enzymes provide. This specificity for substrates is often exquisite and allows enzymes to discriminate between stereoisomers, between substrates that differ by a single functional group, or even between substrates that differ by a single methyl group. For example, tyrosyl-tRNA synthetase (tyrosine-tRNA ligase) distinguishes by a factor of 105 substrates differing by only a single hydroxyl group (Tyr METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and effective strategy in synthetic organic chemistry. New transformations based on known or new enzymes will continue to be exploited. It is worth noting that the number of enzymes reported so far (about 2300) represents only approximately 2% of the total number of enzymes existing in nature. Many unknown enzymes remain to be explored. Known enzymes or proteins can be altered through site-directed mutagenesis or chemical or biological modification to provide new catalytic activities. New protein catalysts specific for a predetermined reaction are now available through the catalytic antibody approach. Multiple enzyme systems required for efficient synthesis of complex molecules such as antibiotics may be constructed within a cell via genetic manipulation and metabolic pathway engineering. It seems fair to say that virtually all kinds of protein catalysts can be constructed from the 20 common amino acids. Figure 14 illustrates our strategy for the development of a specific and efficient protein catalyst for the transformation of a desired reaction. Acknowledgments This work was supported by the National Institutes of Health (GM44154). We thank Professor Richard Lerner for advice on the work involvingFab libraries.
[2 7] M o d i f i c a t i o n o f E n z y m e C a t a l y s i s b y E n g i n e e r i n g Surface Charge
By GREGORIO ALVARO and ALAN J. RUSSELL Introduction The rational modification of enzyme catalysis has been an elusive goal of biochemists for many years.l Until the combination of the techniques of recombinant DNA methodologies and DNA sequencing enabled "protein engineers" to introduce specific changes in the amino acid sequence of proteins via site-directed mutagenesis,2 the alteration of enzyme characteristics was limited to chemical modification? Chemical alterations were rarely specific and often resulted in significant changes in protein structure. Ulmer's insightful review I in 1983 described the properties of enzymes which could be altered in order to "improve" biocatalyst properties, and l K . M. U l m e r , Science 219, 666 (1983).
2 M. J. Zollerand M. Smith,D N A 3, 479 (1984). 3A. Gounaris and M. Ottensen, C. R. Tray. Lab. Carlsberg 35, 37 (1965). METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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almost all these goals have now been reached. Site-directed mutagenesis is now a relatively straightforward procedure and is a common step in the analysis of cloned genes. The term "protein engineering" is also generally accepted to refer to the systematic replacement of amino acid residues in order to alter the properties of a protein. The importance of electrostatic interactions in proteins has been recognized for many years and has been excellently summarized by Warshel et al.4: " T o express the structures of proteins with their functions one must first be able to express structures in terms of energies. Probably the most important requirement for such a correlation is the ability to evaluate the energies of charges in proteins." The key role of surface charge in fine tuning the activity, specificity, stability, and pH dependence of enzymes has been the subject of extensive research since the early 1920s. LinderstrCm-Lang 5 and Kirkwood 6 and colleagues performed pioneering research in developing our understanding of the effect of surface charge on surrounding ionizable amino acid residues. There are currently two approaches to determining quantitatively the importance of charge-charge interactions in proteins: experimental and theoretical. This chapter is concerned with an experimental, rational method for the determination and manipulation of electrostatic effects in enzymes. It is important, however, to realize the limitations of both theory and experimentation in this field. Experimental predictability will result from an accurate theory describing electrostatic interactions, whereas an accurate theory will depend on the existence of experimental systems to test current models. Thus, the most information will be derived from analyses of extensively characterized systems, for which a structure has been determined, and from which the effect of a charge alteration can be measured without interference from conformational changes or altered ionizations. Since the activity and specificity of proteins is so dependent on electrostatic interactions, it is not surprising that protein redesign strategies have been directed at manipulating these types of interactions. Alteration of Surface Charge rather than Internal Charge The distribution of charged groups in proteins is such that over 95% of the charged groups are on the surface (accessible to solvent) and only 5% buried. 7 Furthermore, internal buried charges are usually critically 4 A. Warshel, F. Sussman, and G. King, Biochemistry 25, 8368 (1986). 5 K. LinderstrCm-Lang, C. R. Tray. Lab. Carlsberg 15, 70 (1924). 6 j. G. Kirkwood and F. H. Westheimer, J. Chem. Phys. 6, 506 (1938). 7 D. J. Barlow and J. M. Thornton, Biopolymers 25, 1717 (1986).
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important either catalytically, structurally, or both. For this reason any alteration of internal charge via mutagenesis would have unpredictable results. Surface charge, however, is readily amenable to chemical and site-specific mutagenesis. The first experimental tests of theoretical models for electrostatic interactions utilized chemical modification of charged surface residues to neutralize or reverse the charge on the surface of a variety of enzymes. 3'8-~4 Although chemical modification had significant effects on enzyme function, it was found to be an unpredictable method: similar changes in net surface charge in different enzymes had very different effects on activity and pH dependence) Even so, in 1980 Rees 15 investigated the effect of surface charge on the redox potential of cytochrome c, demonstrating that surface charge could be responsible for a significant portion of the redox control of such proteins. These results were criticized because Rees altered surface charge using chemical modification,~6 resulting in possible conformational changes or artifactual results. What remained clear was that alterations in surface charge resulted in somewhat predictable changes in the redox potential of the protein. Since, when designing an experiment to modify catalysis, it is important to be able to predict the approximate magnitude of any effect resulting from a change in protein structure, chemical modification of surface charge is not the method of choice. The more refined tool of site-directed mutagenesis enables the introduction or removal of charge at a defined point in a protein, allowing ultimate control over which point charges should be altered. Thus, the question now becomes what charge should be altered, and how much of a change in activity would be expected from such an alteration. L o n g R a n g e versus S h o r t R a n g e
There are basically two types of electrostatic interactions within proteins: long range (>5/~) and short range ( Ser-99 would minimize any large conformational changes, whereas the net surface charge would be altered. If data regarding sequence similarities are not available, there are still some useful guidelines which will minimize unexpected and uninterpret-
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able results. First, the side chain which is inserted should be smaller than the one which is being removed. In general, it is far better to leave a hole in the protein after mutation rather than introduce large side chains which require tertiary structural changes in order to accommodate the new amino acid. Although changes on the surface of an enzyme will be less invasive than in the interior, it is still advisable to exercise caution in choosing the mutation to make. Second, if possible it is advisable to use structurally similar amino acids when designing mutations; such alterations are often described as "conservative." For instance, replacing glutamate (or even histidine) with glutamine would be unlikely to result in large conformational changes. Finally, the introduction of amino acids which could distort the backbone of the protein should also be avoided (in particular, proline and glycine residues). Selection o f Mutagenesis Method The general strategy of site-directed mutagenesis is shown in Fig. 1 and has been described previously. 2 The synthetic mutagenic oligonucleotide (previously phosphorylated) is annealed to the viral M13 template DNA containing the gene to be mutated. A DNA polymerase (Klenow fragment, lacking the proofreading function) is then used in in vitro complementary strand synthesis, and DNA ligase is used to ligate the nascent chain to the end of the mutagenic oligonucleotide, forming a partial heteroduplex. This DNA is then used to transfect competent Escherichia coli, giving rise to plaques with either the mutant or wild-type gene. The mutant plaques must then be identified by either colony-blot hybridization, z5 direct plaque screening, or sequencing. After selection of mutant clones, the gene must be fully sequenced to ensure that only the desired mutation has been introduced. A serious but common error in mutagenesis is to sequence the region of the mutation, rather than the entire gene. The efficiency of mutagenesis is affected by a variety of factors. One of the most important of these is the ability ofE. coli to repair the mismatch which has been introduced during mutagenesis. If the two strands could not be distinguished by the host cell then there is a 50% chance that the repair process will correct in favor of the mutant strand. However, M13 DNA synthesized in vivo will be methylated by the enzyme deoxyadenosine methylase, which methylates adenosines in a GATC sequence. Mismatch repair enzymes use this methylase activity in proofreading, 26 and after DNA replication in vitro the newly synthesized strand will be under25 R. B. Wallace, M. J. Johnson, T. Hurose, T. Miyaka, E. H. Kawashima, and K. Itakura, Nucleic Acids Res. 9, 879 (1981). 26 A. Lein-Lu, S. Clark, and P. Modrich, Proc. Natl. Acad. Sci. U.S.A. 82, 7840 (1983).
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Mutagenic Oligonucleotide
L
DNA Polymerase(Klenow) dNTP's DNA Ligase
I Transform Screen Select
FIG. 1. General strategy for site-directed text.
mutagenesis. F o r
a detailed explanation, see
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methylated. Thus, the host cell can distinguish between the parent and daughter strand in favor of the parent. To increase the efficiency of mutagenesis (which is necessary to replace hybridization screening with sequencing), many of the strains used in mutagenesis strategies are deficient in mismatch repair functions (mutL mutants27). Experimental Procedures
Site-Directed Mutagenesis There are now many site-directed mutagenesis techniques which are relatively straightforward. Indeed, once a gene has been cloned into the bacteriophage MI3, it is possible to purchase kits which guarantee mutagenesis efficiencies in excess of 50%. Rather than presenting all the alternatives (these have been described in detail elsewhere28), we discuss the most important factors governing mutagenesis yields, and a typical strategy is outlined. The primary factor governing the success of a mutagenesis experiment is the quality and choice of oligonucleotide. In particular, low-purity oligonucleotides or otigonucleotides which do not bind specifically to the template DNA can result in extremely low yields of mutants. Care must be taken when designing oligonucleotides to ensure absolute specificity of binding. It is advisable to check the oligonucleotide in a sequencing reaction for sequencing capability prior to use. This gives a good indication as to whether the oligonucleotide is pure and specific enough to be used. Once pure template has been obtained as described previously,27 the mutagenesis reaction sequence is initiated with the annealing of oligonucleotide and template. The purity of template is as important as that of the oligonucleotide. Annealing. One hundred picomoles of primer (oligonucleotide) is kinased by incubating the following mixture for 30 min at 37° and then 10 min at 70 °. 10/zl Primer 2 /zl 10 × KB buffer [500 mM Tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.5, 100 mM magnesium chloride, 50 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM spermidine hydrochloride] 1/zl Dithiothreitol (100 mM) 27 p. Carter, H. Bedouelle, M. M. Y. Waye, and G. Winter, "Oligonucleotide Site-Directed Mutagenesis in M I 3 . " Anglian Biotechnology Limited, 1985. 28 R. J. Leatherbarrow and A. R. Fersht, Protein Eng. 1, 7 (1986).
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2/zl Adenosine triphosphate (10 mM) 4/zl Sterile water 1/xl T4 polynucleotide kinase (0.5 units//xl) One microgram of M13 template DNA should then be added to 10 pmol of kinased primer in a final volume of 10 t-d (1 x TM: 100 mM Tris-HC1, pH 8.0, 100 mM magnesium chloride), placed in a beaker of hot water (80°), and allowed to cool to room temperature ( - 2 0 min). Extension and Ligation. To the 10/zl of annealing mix, the following should be added: 1/xl 10 x TM buffer 1 /xl Deoxynucleotide triphosphates (5 mM each of adenosine, guanine, cytosine, and thymine) 1/zl Dithiothreitol (I00 mM) 4/~1 Sterile water 1/xl T4 DNA ligase (10 units//zl) 0.5/xl DNA polymerase, Klenow fragment (5 units//xl) Incubation is conducted overnight at 15° before the mixture is used to transfect E. coil as described previously. 29 Plaque Screening. Plates containing approximately 250 plaques can then be used in direct plaque screening, if the transformations have been performed using H-Top agarose instead of H-Top agar. Place a circular nitrocellulose filter disk on the plate; remove the disk after 5 min, and bake the filter for 2 hr. The filter should then be prewetted in 6 x SSC buffer [from a 20 x SSC stock (17.5% (w/v) sodium chloride, 8.8% (w/v) sodium citrate, pH 7.0)] for 5 min in a sealed plastic bag and prehybridized at 67 ° for 2 hr. After rinsing the filters in 6 x SSC, they should be placed in another plastic bag with 10 pmol radiolabeled mutagenic oligonucleotide, incubated at 67 ° for 30 min, and allowed to cool to room temperature overnight. The filters should then be washed in buffer, dried, and autoradiographed. Starting at 5° below the melting temperature for the oligonucleotide (as calculated from Wallace's rules29), this cycle of washing and autoradiography should be performed at increasing temperatures (with 2° increments) until mutant and wild-type colonies can be distinguished. Once mutant plaques have been identified, they can be used in preparation of template DNA for sequencing 3° and eventually subcloning into an ex29 S. V. Suggs, T. Hirose, T. Miyake, E. H. Kawashima, M. J. Johnson, K. Itakura, and R. B. Wallace, in "Developmental Biology Using Purified Genes" (D. Brown, ed.). Academic Press, New York, 1981. 30 A. T. Bankier and B. G. Barrell, in "Techniques in the Life Sciences B5, Nucleic Acids Biochemistry, B508." Elsevier, Ireland, 1983.
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pression vector. Expression and purification of the protein will enable characterization of the effects of the electrostatic alterations. Techniques such as casette mutagenesis 31 and the polymerase chain reaction 32 have not been discussed here, but they will undoubtedly impact greatly on the ease of mutant protein screening and production. The availability of mutagenesis kits has led to a dramatic reduction in the time necessary to make mutants. However, the analysis of each mutant is the critical part of determining structure-function relationships in proteins, and the temptation to produce large numbers of mutants, without a detailed investigation of the properties of each, must be resisted. Importance of Plaque Purification and Sequencing. Once a plate containing many plaques has been generated, there is a temptation to perform template DNA preparations for sequencing immediately. This will often lead to a mixed population of template DNA, since each plaque can contain both wild-type and mutant DNA (as a result of bacteriophage migration in the plate). Another unfortunately common practice is to sequence only the region of DNA surrounding the site of mutation. It is absolutely necessary to sequence the entire coding region of the protein if interpretation of the data is to be valid.
Characterization of Mutant Proteins In order to present a meaningful explanation for the properties of mutant proteins with altered surface charge, the wild-type and mutant proteins should be subjected to extensive kinetic (and, if possible, structural) investigations. Although the methods used in characterizing mutant proteins will be different for each family of enzymes (and are beyond the scope of this chapter), the following section provides comments which are important for the analysis, and interpretation, of all systems. Need for Active-Site Titration. Of utmost importance is the need for a pure sample of the mutant protein, free of contamination from wild-type or other mutant proteins. If a mutant protein has reduced activity relative to wild type, then the presence of small amounts of wild-type protein could result in a false interpretation of the activity of a mutant. The most simple way to determine if the activity present in a sample of mutant enzyme is the result of only a small fraction of the total protein is with an active-site titration. Much of the work in the area of protein engineering of surface charge has been performed with proteases, and quantitative knowledge of the concentration of all reacting species is particularly important because of the problem of autolysis. Titrating enzyme with a variety of small 31 j. A. Wells, M. Vasser, and D. B. Powers, Gene 34, 315 (1987). 32 H. A. Ehrlich, " P C R Technology." Stockton Press, New York, 1989.
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organic molecules avoids the internal inconsistency of using a rate assay to calibrate a method which uses a rate assay as its absolute standard. The organic active-site titrants have the property that their reaction with the enzyme is dependent on the integrity of the active center. Thus, the titration depends not on the enzyme itself, but rather on the substrate. For instance, the protease subtilisin can be titrated with N-trans-cinnamoylimidazole as described in detail previously. 33 Unfortunately, activesite titrations are not available for most enzymes, although an assay could be developed if a tight-binding competitive inhibitor can be identified. In such cases, reacting the enzyme with radiolabeled inhibitor and recovering the complex would enable the calculation of the concentration of enzyme from the amount of radiolabel present after the removal of unbound titrant. The active-site titration of tyrosyl-tRNA synthetase (tyrosine-tRNA ligase), the first enzyme to be subjected to site-directed mutagenesis, is performed in such a way and has been described previously. 34 Finally, it is important to point out that a careful kinetic investigation of mutant and wild-type enzymes will be meaningless in the absence of good data analysis techniques. In particular, the use of linear regression to solve Lineweaver-Burke plots (which enable the "determination" of kcat and Km is totally unacceptable. There are now a number of excellent computer programs (which are as user-friendly as a calculator) that perform nonlinear regression on the original data. 35 Nonlinear regression of data does not distort the experimental error in kinetic determinations and should be used in all instances. In general, computational analysis of data is faster and more accurate than fitting by eye (which is still performed in some laboratories), but it is always necessary to understand the limitations of the computer and to remember that a computer will fit even the worst data to a given equation. Dielectric Constant and Effect of Ionic Strength. This chapter suggests that by altering surface charge it is possible to manipulate biocatalytic behavior in a rational manner. Such a method depends on the strength of electrostatic interactions between the surface of a protein and the amino acids that are directly involved in binding and catalysis, which in molecular terms are far from the site of mutation. The strength of an electrostatic
33 M. L. Bender, M. L. Begoue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kezdy, T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966). 34 A. R. Fersht, J. P. Shi, J. Knill-Jones, D. M. Lowe, A. J. Wilkinson, D. M. Blow, P. Brick, P. Carter, M. M. Y. Waye, and G. Winter, Nature (London) 314, 235 (1985). 35 " E N Z F I T T E R " and " G R A F I T " were written by R. J. Leatherbarrow (Imperial College, London), and are specifically designed for enzyme data.
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
633
interaction will depend on the ability of the material separating the charges to attenuate the interaction. For homogeneous materials the ability of a material to polarize an electric field is described by the dielectric constant (D). Free space, by definition, has a dielectric of I, while pure water has a dielectric of 78.5 at 25 °. D is a macroscopic quantity which varies inversely with temperature. When small diffusible ions are included in a solvent, the dielectric for the solution increases and is inversely related to the separation distance between two point charges in the medium. Hill 36 defined this Debye-Htickel screening 37 as an effective dielectric constant (Deer), which is related to the dielectric of water (Dw) as follows:
Deer = D w ( e kr) where k is the Debye-Hiickel parameter, which is proportional to ionic strength, and r is the separation distance between the point charges. Although the applicability of the concept of dielectric constants to proteins is discussed later, there is no question that the strength of an electrostatic interaction will depend on the ionic strengh of the solvent. Thus, any modification of enzyme properties as a result of alteration of surface charge should also be dependent on ionic strength: increasing ionic strength should decrease the effect of the mutation. Testing the effect of ionic strength on the difference between mutant and wild-type proteins is necessary for protein engineering strategies which depend on the alteration of surface charge. Figure 2 shows the dependence of the difference between the PKa of a surface charge mutant of subtilisin and wild-type subtilisin on ionic strength. 38 Note that as ionic strength increases, the effect of the mutation becomes negligible. If such a test does not demonstrate a dependence on ionic strength, it is unlikely that the effect of the mutation is a result of an altered electrostatic interaction. From the preceding discussion it is obvious that the effect of a change in surface charge will be maximized at low ionic strengths, and experiments should be designed with this in mind. Experimental Determination of Dielectric Constants in Proteins. As pointed out above, the strength of electrostatic interactions in proteins will be a function of the dielectric constant in the region which separates the charges. Thus, in order to predict the effect of a change in surface charge on a distant charged amino acid, the dielectric constant must be determined. In the same way that the strength of hydrogen bonds 34 and 36 T. L. Hill, J. Phys. Chem. 60, 253 (1956). 37 p. Debye and E. Hiickel, Phys. Z. 24, 185 (1923). 38 A. J. Russell and A. R. Fersht, Nature (London) 328, 496 (1987).
634
ENZYMES
[27]
0.5
~"
0.4
03 I
0.3 4-) "o ,-.t -~
0.2
Q., 0.1
0.0 0.001
0.005
0.01
Ionic
0.025
Strength
0.1
0.5
1
(M)
FIG. 2. Dependence of ApKa of histidine-64 between wild-type and DS99 subtilisins on ionic strength. The Ap Ka values represent the difference in PKa values for the pH dependence of kcat/K m between wild-type and aspartate ~ serine-99 (DS99) subtilisins in the hydrolysis of succinyl-L-alanyl-L-alanyl-L-prolyl-t-phenylalanyl-p-nitroanilide. The assays were performed at the ionic strengths indicated, as described previously [A. J. Russell, P. G. Thomas, and A. R. Fersht, J. Mol. Biol. 193, 803 (1987); P. G. Thomas, A. J. Russell, and A. R. Fersht, Nature (London) 318, 375 (1985)]. Note that the 0.001 M experiments utilized imidazole buffers, whereas the remaining experiments were performed with phosphate buffers.
hydrophobic interactions 39 have been measured by their removal through mutagenesis, the energy of an electrostatic interaction can also be calculated from the effect of its removal. As for any determination of energies from differences between mutants, these free energies are apparent free energies representing the sum of one or more energy changes. Thus, the effect of a small perturbation in enzyme structure which removes (or implants) an electrostatic interaction will enable the calculation of the apparent free energy of that particular interaction. In addition, if the kinetic effect of the mutation can be localized to an effect on one particular charged residue, and the distance separating the surface point charge and 39 j. T. Kellis, K. Nyberg, D. Sali, and A. R. Fersht, Nature (London) 333, 784 (1988).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
635
that residue is known, then an apparent dielectric constanP ° for the region between these charges can be estimated. By systematically replacing and introducing charged surface residues, it should be possible to map the dielectric constant through different regions of the protein. The temptation is to use one value of dielectric for the whole protein, since this simplifies the system considerably, but this type of uniform continuum electrostatic model is obviously a gross oversimplification of reality. Also, in terms of an experimental determination of dielectric for a particular interaction, it is crucial to remember that this value will refer only to that specific interaction and should not be used to describe the whole protein. In referring to the dielectric properties of proteins Rogers 41 has likened proteins to Swiss cheese, that is, there appear to be lines of hydrophilicity (and hence high dielectric) within the generally hydrophobic interior of globular proteins, demonstrating the ease with which data can be overinterpreted. In addition, as Warshel and Russell 42 have pointed out, charged groups do not only interact with each other, but also with the protein dipoles. Calculations using experimental data should take into account these interactions wherever possible. With all these restrictions and caveats in mind for the interpretation of experimental data, there may seem little point in calculating a value of the dielectric. However, without knowledge of the screening of charges within a protein and at the protein-solvent interface, understanding of the electrostatic properties of proteins cannot develop. The term apparent dielectric constant has been proposed 4° to describe dielectric constants calculated from mutant protein data. This term stresses the local nature of dielectrics within a protein, while at the same time retaining the concept of a dielectric constant. Control Mutations. The mere confirmation that an enzyme has altered properties as a result of a change in surface charge does not conclusively prove that the effect is a result of a modified electrostatic interaction. In order to avoid misinterpretation of data, it is advisable to repeat a surface charge alteration at a site distant from that of the first mutant. In addition, "control" mutations should be generated at each site. For instance, if an aspartate to serine mutation is introduced into the subtilisin sequence at position 99 (see Table I), a suitable control mutation would be the introduction of a lysine at the same position (thus doubling the change in net surface charge). The need for control mutations is particularly ira-
4o A. J. Russell, P. G. Thomas, and A. R. Fersht, J. Mol. Biol. 193, 803 (1987). 41 N. K. Rogers, Prog. Biophys. Mol. Biol. 48, 37 (1986). 42 A. Warshel and S. T. Russell, Q. Rev. Biophys. 17, 283 (1984).
636
ENZYMES
[27]
portant given that the strategy outlined herein will generate small, but measurable, changes in enzyme function. Ideally, the structure of each mutant should be determined in order to show that alterations in enzyme properties are not due to fortuitous changes in protein structure. Where this is not possible, further mutations and detailed kinetic analysis will aid interpretation of the results. For instance, mutation of charged surface residues could damage metal ion binding sites (in proteases these are particularly important for stabilizing the enzyme). Kinetic analysis of the stability of mutants in the presence and absence of such metal ions will indicate whether a change in activity is an indirect effect of an alteration in metal binding.
A p p l i c a t i o n s of S u r f a c e C h a r g e M o d i f i c a t i o n for A l t e r i n g Enzyme Properties
pH Dependence, Specificity, Stability, and Activity The first use of site-directed mutagenesis to tailor the properties of enzymes by modification of surface charge was directed at altering the pH dependence of subtilisin. 43 Mutant subtilisins were generated with shifted pH-activity profiles, higher catalytic activities, and altered specificities. Subtilisin is a serine protease which can catalyze the hydrolysis of amide or ester bonds via the acyl-enzyme mechanism. Because subtilisin is so well characterized both structurally and mechanistically, it has been described as an enzyme "designed to be engineered."44 During catalysis the imidazole side chain of His-64 acts as a general base. Thus, subtilisin is only active when His-64 is in the unprotonated state, and the activity of the enzyme follows exactly the pKa of this residue; in other words, the pK a of the enzyme (as can be measured via the pH dependence ofkcat/Km) is equivalent to the macroscopic pKa of His-64. Since the protonated form of His-64 will be stabilized by negative charges (even those distant from the active site), the neutralization of such charge should increase activity at low pH, and thus decrease the PKa of the enzyme. This hypothesis was tested using site-directed mutagenesis. The mutations introduced were at surface residues 99 and 156 (both of which have negatively charged side chains in the wild-type enzyme) of subtilisin BPN'. In each case the amino acid was replaced by a serine (with a neutral side chain) and a lysine residue (with a positively charged side chain). The effect on the pH dependence of catalysis was studied by following the pH 43 p. G. Thomas, A. J. Russell, and A. R. Fersht, Nature (London) 318, 375 (1985). 44j. A. Wells and D. A. Estell, Trends Biochem. Sci. (Pers. Ed.) 13, 291 (1988).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
637
1.2_
1.0
?i!i;i!i!i!i!i!i:?!i~i~ ~i'i:i:i~:;i!i:i;iii;i': i~i:i:i~i:iii?i::?:: iiii:i~i~i:i~iii:i:::i'i ii;i;ii;iiiiiii?:~i;3~ !iiii?ii~;:ii;:.~ii;i iii:?:iiiiiii:;ii:i-iiii ::::::::::::::::::::: :::::::::::::::::::::::
0.8
?!i~i:i!ili:i!i~??:'i iiii?i:iii~i:i!i~i:i:
""
i!i!i!i!i!ili!i!i!i!i!i!!!i!!!
0.6
!i!i;i!i;i~i~-~iiii-iiii !i!iii:iiiiii:iii-?iiii
::::::::::::::::::::::::::
??????i~?i????:i
0.4
ii!iiiiiiii:i-iii~L:i
ii:ilii~ili;:i;i;:i:ili
:::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::::
!i~i!i!i:i!i!i~i~ili~!~;ii~ii!
::::::::::::::::::::::::::::
i!i!iii:i~i!i!i!i~i!i i~!ii~ii ::::::::::::::::::::::::::::
i:i:ii?:i:: :::: i3:i:?i:?:: ?ii: ::i:i: i ?i~i:i ?i
i~iiiii?i~iiii!ili!i!i~ii:~ii iiiiiiiiiiiiii
::::::::::::::::::::::::::::::
::::::::::::::::::::::::::::
0.2
iiiiiiiiiiii!i~i!iiiiiii)iiii; iiiiiiii!iiiiiiiiiiiiiiili!iii
:::::::::::::::::::::
::::::::::::::::::::::::::::::: :i:i:iii!iiiii!i!!~iii!!iiiiii :+:+:+x+>::+:+ x+:+x:+:+>:+: :::::::::::::::::::::::::::::: :i:i:?:i:i:i:i•i-i-i:i•3!:i:i :::::::::::::::::::::::::::::
:::::::::::::::::::::::::::::::::::::::::
0.0
|
D-$99
|
E-S156
|
i
E-S,D-S
D-K99
E-K156
ENZYME FIG. 3. Shift in the pK a of histidine-64 of mutant subtilisins, relative to wild type, at 0.001 M ionic strength. The APKa values given are for the pH dependence of keat[K m in the hydrolysis of succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-p-nitroanilide. The assays were performed at 25 ° in imidazole-imidazolium buffer (ionic strength 0.001 M). The naming of the enzymes is such that the first letter represents the residue which has been mutated to a different amino acid (given by the second letter) at a particular position (given by the number).
dependence of kcat/Km; this measures the apparent PKa of the active-site histidine (His-64). In contrast to earlier studies using chemical modification to alter pH dependence which showed relatively small shifts, removal of just one surface carboxylate in subtilisin (Asp-99) lowers the pKa of its active-site histidine by 0.3 units, despite the groups being approximately 1.3 nm apart. Similar results were obtained on removal of a negative charge at Glu-156, which is approximately 1.4 nm away from His-64. These shifts in pKa are cumulative on sequential or simultaneous changes in charge. Double mutants show increased effects, as do single mutants which have a double charge change. Indeed, in the case of a quadruple charge change, the PKa of activity is decreased by 1 pH unit (see Fig. 3). The shifts in pK~ are
638
ENZYMES
[27]
ionic strength dependent (as shown in Fig. 2), as would be predicted for long-range electrostatic interactions. The detailed kinetic investigation of these mutant enzymes has also given insight on dielectric properties at the protein surface, the role of ions in electrostatic shielding, and field effects on catalysis. From the changes in pH dependence of catalysis it has been possible to determine the magnitude of an electrostatic interaction between two point charges, as well as to determine the effect of these mutations on specificity and activity. In addition, counterion binding to charged groups was shown to be important when interpreting kinetic data. The shifts in pK a of the mutant enzymes enable the calculation of the apparent free energy of interaction between His-64 and the mutated residue (AAG). It is particularly important to point out that this energy is calculated from the removal of an interaction, and thus is influenced by other factors. This is analogous to the calculation of the apparent free energy for a hydrogen bond using protein engineering. 34 These values for AAG are in the region of 0.5 kcal/mol for the interaction of two charges separated by approximately 1-1.5 nm. The analogous value for a hydrogen bond, over a distance of 0.3 nm, is approximately 1 kcal/mol (depending on the type of hydrogen bond). These values demonstrate the great importance of electrostatic interactions in proteins. From determinations of AAG it was possible to calculate the apparent dielectric constant for the region separating the two chargesY These calculations are subject to even more assumptions than those involved in estimating the original interaction energy. Although the calculations are an oversimplification, the results indicate that the dielectric constant in the region of the active-site cleft of subtilisin is between 40 and 50, approaching that of water. Furthermore, the relationship between measured dielectric and ionic strength fits the relatively simple Hill model 36 for electrostatic interactions. This does not mean that the Hill assumptions are correct, but rather that the data can fit the approximation, lending credence to the high value of dielectric observed. Finally, it is important to reemphasize the warning of Hill, namely, that the effective dielectric constant between any two points in a system such as a protein is a unique value for those two points and is not applicable to any other pair of points. Although these mutants were not designed to alter specificity and activity, a number of interesting correlations were observed. Glu-156 is located close to the substrate binding site of subtilisin, and as would be predicted the removal or reversal of negative charge at this position
45 N. Bjerrum, Z. Phys. Chem. Stoechiom. Verwandtschaftsl. 106, 219 (1923).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
639
increases the affinity of the enzyme for negatively charged substrates while decreasing the affinity for positively charged ones. Indeed, this was the first demonstration of the control of substrate specificity by alteration of distant surface charge. The study of subtilisin has shown that, as predicted by the classic LinderstrCm-Lang theory, 5 making the surface charge of a protein more negatively charged raises the pK a of acidic groups (they lose a positively charged proton on ionization). Conversely, making the surface more positively charged lowers the pKa of surrounding acidic groups. Since these changes are due to electrostatic effects, they will be maximized at low ionic strengths. Significant effects will, however, be manifested at ionic strengths as high as 0.1 M if multiply charged counterions are avoided. Indeed, mutations should be designed such that they do not concentrate counterions in the active-site cavity. It would be foolish to assume that all systems will be as predictable in their response to surface mutations as subtilisin has been. This work has, however, shown again the importance of long-range electrostatic interactions, and how via their manipulation the properties of a protein can be significantly and predictably altered. The pH-activity dependence of lysozyme has also been predictably altered by engineering of surface charge .46 In this instance the enzyme was redesigned to be a more efficient catalyst than wild type under conditions of high pH and ionic strength (the mutation used introduced positive charge at only two positions on the surface). In addition, the removal of positive charge at two surface residues enabled the generation of a mutant enzyme which could function at low pH and ionic strength. Removal and introduction of surface charge has also been used to test structure-function relationships in an enzyme. Watson and colleagues 47 have shown that an arginine residue on the surface of phosphoglycerate kinase is important for catalysis, even though it is situated 1.3 nm from the active site. This has been used to infer that, during catalysis, a large conformational change takes place which will bring the arginine into close proximity with the active site. Mutation of surface residues has also led to important advances in the analysis of charge effects on protein-protein interactions. 48'49 Naturally,
46 M. Muraki, M. Morikawa, Y. Jigami, and H. Tanaka, Protein Eng. 2(1), 49 (1988). 47 p. A. Walker, J. A. Littlechild, L. Hall, and H. C. Watson, Eur. J. Biochem. 183, 49 (1989). 48 j. E. Long, B. Durham, M. Okamura, and F. Millet, Biochemistry 28, 6970 (1989). 49 p. C. Weber, T. J. Lukas, T. A. Craig, E. Wilson, M. M. King, A. P. Kwiatkowski, and D. M. Watterson, Proteins: Struct. Funct. Genet. 6, 70 (1989).
640
ENZYMES
[27]
interactions between proteins occur at the surface, and thus the role of charged amino acids will be significant. It is important to realize, however, that these interactions are generally short range. Charged surface residues which are located close to the substrate binding site of subtilisin have also been used to design substrate specificity in subtilisin, s° Long-range electrostatic interactions are also important when considering the stability of proteins. Indeed, it has been shown that such interactions influence the stability and cooperativity of dihydrofolate reductase.51 Finally, surface charge mutations have also been used to dissect the charge effects in protein-metal binding: 2 ion transport: 3 ion binding, 54 and isolated helical structures) 5 Solvent Resistance
The surface amino acid side chains of proteins are not only important in fine tuning the electrostastic nature of the active site (as demonstrated by the work described above), but they also perform the central role in protein-solvent interaction. Solvent structure is of such importance that in order to interpret mutagenesis results completely, alterations in the organization of bound water molecules must be considered. This is of particular interest when considering strategies designed to enable proteins to function in low water (the typical solvent for enzymes) environments. There are several factors that necessitate the use of organic solvents as media for enzyme-catalyzed reactions. 56 First, most hydrophobic reagents of interest to synthetic chemists are soluble only in organic solvents. Second, an organic bulk solvent can shift the thermodynamic equilibrium of many hydrolysis reactions (e.g., of esters and peptides) to synthetic ones. Third, the use of organic solvents obviates the danger of bacterial contamination, which is a major problem in many aqueous biocztalytic reactors. Because such an approach is attractive, the field of enzyme
50 j. A. Wells, D. B. Powers, R. R. Bott, T. P. Graycar, and D. A. Estell, Proc. Natl. Acad. Sci. U.S.A. 84, 1219 (1987). 51 K. M. Perry, J. J. Onuffer, M. S. Gittleman, L. Barmat, and C. R. Matthews, Biochemistry 28, 7961 (1989). 52 S. R. Martin, S. Linse, C. Johansson, P. M. Bayley, and S. Fors~n, Biochemistry 29, 4188 (1990). 53 R. MacKinnon, R. Latorre, and C. Miller, Biochemistry 28, 8092-8099 (1989). 54 S. Linse, P. Brodin, C. Johansson, E. Thulin, T. Grundstrom, and S. Fors6n, Nature (London) 335, 651 (1988). 55 D. Sali, M. Bycroft, and A. R. Fersht, Nature (London) 335, 740 (1988). 56 A. Zaks and A. J. Russell, J. Biotechnol. 8, 259 (1988).
[27]
MODIFYING CATALYSIS BY ENGINEERING SURFACE CHARGE
641
catalysis in organic solvents (first reported in the 1960s 57) has undergone rapid expansion, particularly since the mid 1980s. The use of enzymes in organic solvents is becoming the norm for applied enzymologists and even synthetic organic chemists, 58 and naturally there is much interest in designing proteins with improved properties for use in hydrophilic organic solvents. There is little question that some water is an absolute necessity for the maintenance of the catalytic integrity of enzymes. Indeed, enzyme structure and function rely on the direct and indirect role of water in all the noncovalent interactions. 59 Although the need for water is obvious, the amount of water required in enzymatic systems is less clear. The estimation of the thickness of the water layer around the enzyme is subject to speculation because the minimum amount of water necessary to allow enzymes to function has been determined for relatively few enzymes. 6° The hydration of proteins consists of a number of distinct stages. For lysozyme, water molecules first gather around charged groups and then cluster and grow over most of the protein surface. When only half of the protein surface is covered in water molecules the enzyme is partially active. Thus, the total amount of water necessary for lysozyme activity is relatively low as long as the water is associated with the enzyme surface. It is the concentration of water on the enzyme which determines specific activity, and it has been proposed that the major factor in determining activity in nonaqueous media stems from the ability of a particular solvent to strip water from the enzyme. 6~ Importantly, there is no question that the amount of water required for optimal activity in organic solvents is enzyme-specific. This phenomenon means that some enzymes are "better" than others in organic solvents in that they can function in very low water environments in hydrophilic media. For instance, subtilisin is the only enzyme known which can function in anhydrous dimethylformamide. It seems obvious that proteins with modified behavior in organic solvents will have altered surface charge, since it is the charged surface residues which interact most strongly with solvent. It has been suggested that the best method to achieve stability of a correctly folded protein in an anhydrous environment is with the use of a set of "design rules. ''62 57 F. R. Dastoli and S. Price, Arch. Biochem. Biophys. 122, 289 (1967). 58 j. B. West, W. J. Hennen, J. L. Lalonde, J. A. Bibbs, Z. Zhong, E. F. Meyer, and C.-H. Wong, J. Am. Chem. Soc. 112, 5313 (1990). 59 G. E. Schulz and R. H. Schimer, in "Principles of Protein Structure." Springer-Verlag, New York, 1979. 6o j. A. Rupley, E. Gratton, and G. Careri, Trends Biochem. Sci. (Pers. Ed.) 8, 18 (1983). 61 A. Zaks and A. M. Klibanov, J. Biol. Chem. 263, 3194 (1988). 62 F. H. Arnold, Protein Eng. 2, 21 (1988).
642
ENZYMES
[27]
Arnold's design rules predict that the removal of unpaired surface charges will stabilize proteins owing to the energy gained by removal of an unfavorable interaction between a charged group and a relatively low dielectric solvent. This is in agreement with all the electrostatic models mentioned above. There are two problems with this approach that may, however, hinder rapid progress (as has been recognized by Arnold62). If it becomes necessary to remove most of charged groups in order to stabilize the protein, there will be difficulty expressing the new hydrophobic protein. This may not be a significant problem if it turns out that only a few charge neutralizations are necessary to stabilize the protein significantly. The major foreseeable problem will be the effect of the mutations on the activity of the enzyme. As described above, electrostatic interactions (which actually encompass all interactions) throughout the protein will be significantly affected by changes in the dielectric environment of the protein. Thus, it would not be surprising if the removal of surface charge, which would result in a close interaction between organic solvent and protein, were to disrupt the finely tuned electrostatic interactions with control the catalytic mechanisms of most enzymes. Naturally, the experimentation required to test the deisgn rules is being performed by Arnold. 63 An alternative approach (currently being investigated in our laboratory) is to introduce more charged residues onto the surface of the enzyme while keeping the net surface charge constant. This should enable the enzyme to retain a "thicker" layer of water when placed in organic solvents. This layer of water could then act as a high dielectric shield, effectively protecting the protein molecule from bulk solvent. The success of this approach will depend on the relative partitioning of water between an enzyme and a given solvent, and whether a more charged enzyme will shift the partitioning of water in the solvent from that solvent to the enzyme surface. Concluding Remarks The huge potential of protein engineering is demonstrated by the massive proliferation in investigations utilizing this technology since the first experiments of the early 1980s. 64 The ability to alter specifically one or more residues in a protein at will is such a powerful tool that it enables the investigator to determine energies of interaction between amino acid side chains within a protein. This information can then be used in the ab 63 F. H. Arnold, personal communication (1990). 64 G. Winter, A. R. Fersht, A. J. Wilkinson, M. Zoller, and M. Smith, Nature (London) 299, 756 (1982).
[28]
REMODELING
ENZYME
SPECIFICITY
643
initio design of new proteins which could catalyze novel processes. Indeed, the information derived from protein engineering studies has already been utilized in the design of a chemically synthesized " e n z y m e " with chymotrypsin-like properties. 65 The most obvious prerequisite for site-directed mutagenesis is that the enzyme under investigation has been cloned, sequenced, and expressed in a suitable system. The technique is, however, at its most powerful when a detailed knowledge of structure and function is available. Only in the light of such information will mutagenesis experiments yield information on the crucial relationships between structure and function. In many cases it is a relatively simple step to clone and express a given protein, and thus the rate-limiting step for protein engineers is in the analysis of the mutants generated. Acknowledgments Much of the work described in this chapter was performed by Dr. P. G. Thomas in the laboratory of Professor A. R. Fersht, and this review would not have been possible without their contributions. The work was funded by Science and Engineering Research Council, UK. We also thank Dr. F. H. Arnold for helpful discussions about designing solvent-resistant enzymes. The solvent-resistant enzyme project of A.J.R. and G.A. is currently funded by grants from the National Science Foundation (BCS-9057312), the Petroleum Research Fund (Type G: 22019-G4), and the University of Pittsburgh Internal Grant Program. G.A. is a Spanish Ministry of Science and Education Postdoctoral Fellow. 65 K. W. Hahn, W. A. Klis, and J. M. Stewart, Science 248, 1544 (1990).
[28] M u t a t i o n a l R e m o d e l i n g o f E n z y m e S p e c i f i c i t y
By ROGER BONE and DAVID A. AGARD Introduction Limiting the range of substrates which are catalytically productive is one of the fundamental functions that enzymes provide. This specificity for substrates is often exquisite and allows enzymes to discriminate between stereoisomers, between substrates that differ by a single functional group, or even between substrates that differ by a single methyl group. For example, tyrosyl-tRNA synthetase (tyrosine-tRNA ligase) distinguishes by a factor of 105 substrates differing by only a single hydroxyl group (Tyr METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
