Clin. Biochem. 9, (3) 155-159 (1976)
How An Enzyme Works A. H. B L A I R Department
of B i o c h e m i s t r y , D a l h o u s i e U n i v e r s i t y .
Halifax, Nova
Scotia
B3H
4H7
well i l l u s t r a t e the development of this technique. Myoglobin, the f i r s t p r o t e i n for which the threed i m e n s i o n a l s t r u c t u r e was obtained (by Sir John K e n d r e w ) , is relatively small, c o n t a i n i n g only 153 residues. The work could be completed to a resolution of 6 A. before really f a s t computers were available to p e r f o r m the calculations, since only 400 reflections were required. By contrast, a resolution of 1.4 A (the limit of this technique) r e q u i r e d 25,000 reflections. Lactate dehydrogenase from dog-fish muscle cont a i n s a p p r o x i m a t e l y 1300 residues in four d i s t i n c t polypeptide c h a i n s ; solution to the 2.5 ~, level for the M., s u b u n i t r e q u i r e d calculations based on approximately 14,000 terms. Even at a resolution of 2.5 A, not all i n d i v i d u a l a m i n o acid residues can be i d e n t i f i e d with c e r t a i n t y f r o m X - r a y data alone, but require parallel use of the chemically d e t e r m i n e d amino acid sequence.
CLBIA, 9, (3) 155-159 (1976) Clin. Bioche~n.
Blair, A. H. D e p a r t m e n t of B i o c h e m i s t r y , Dalhousie [bHversit~./, H a l i f a x , N.S. B 3 H 4 H 7
HOW AN ENZYME WORKS The structure of enzyme active sites and the naturc of the catalytic process are reviewed. The impressive efficiency of these protein catalysts appears to stem from such factors as proximity and orientation of enzyme and substrate moieties, strain, and the occurrence of distinctive microenvironments within catalytic centres. Carboxypeptidase, lysozyme, and aspartate transcarbamylase, which have been extensively investigated by many techniques, serve tt, illustrate the application of these concepts. r
THIS BRIEF REVIEW ~:oncerns the s t r u c t u r e of enzyme active sites and the n a t u r e of the catalytic process. I t also outlines those factors associated with ligand b i n d i n g t h a t modulate events at an active site - - an aspect crucial to an u n d e r s t a n d i n g of how enzymes work in association with other cellular c o n s t i t u e n t s . To u n d e r s t a n d enzyme catalysis one m u s t relate s t r u c t u r e to f u n c t i o n . The sequence of amino-acid residues in an enzyme protein can be elucidated by wellestablished chemical m e t h o d s ; the t h r e e - d i m e n s i o n a l o r i e n t a t i o n and position of c o n s t i t u e n t residues can be d e t e r m i n e d by X - r a y d i f f r a c t i o n , which maps the e l e c t r o n - d e n s i t y d i s t r i b u t i o n . This t e c h n i q u e requires a protein t h a t will form defined c r y s t a l l i n e complexes with heavy atoms (e.g., H g ) . In recent years, a n u m b e r of enzymes and other p r o t e i n s have been mapped at high resolution (Table 1), and enzymes c o n s i s t i n g of multiple s u b u n i t s are now b e i n g investigated. Two proteins, namely myoglobin and lactate dehydrogenase,
The a m i n o acid residues t h a t are located at the active site (i.e., in a position to i n t e r a c t with the s u b s t r a t e d u r i n g catalysis) can be i d e n t i f i e d t h r o u g h selective chemical modification, by a n a l y s i n g the effect of m o d i f i c a t i o n on catalytic a c t i v i t y . " ' This approach has the a d v a n t a g e of dealing with the enzyme in solution, where conditions more closely a p p r o x i m a t e the physiological s t a t e ; however, lack of specificity in the reaction may complicate i n t e r p r e t a t i o n of results. Several approaches are possible for i d e n t i f y i n g the active-site region in an X - r a y - d e r i v e d s t r u c t u r e . F i r s t , if a t i g h t l y bound prosthetic group is involved in the reaction, this provides a m a r k e r t h a t will be a p p a r e n t in the X - r a y d i f f r a c t i o n p a t t e r n . These groups include metal ions (e.g., zinc in dehydrogenases). Second, and of potentially wider applicability, is the a c t i v i t y of some enzymes in t h e i r crystalline s t a t e : t h a t is, t h e i r s u b s t r a t e diffuses into the crystal to the active site, binds, and undergoes reaction. A very slowly r e a c t i n g s u b s t r a t e may form a
TABLE 1 X-RAY CRYSTALLOGRAPHYOF PROTEINS
Protein
Source
Mol wt
Number of residues
subunits
Resolution 6 2 1.4 5.5 2.8 6
Myoglobin . . . . . . . . . . . . . . .
Whale
17,800
153
--
Oxyhaemoglobin . . . . . . . . . .
Horse
64,500
574
4
Lysozyme . . . . . . . . . . . . . . . .
Hen egg
,
14,600
129
--
Carboxypeptidase . . . . . . . . .
Bovine pancreas
34,600
307
--
Ribonuclease. . . . . . . . . . . . . Ribonuclease S . . . . . . . . . . .
Bovine pancreas
13,683 13,700
124 124
---
Bovine pancreas Dog-fish muscle
25,000 150,000
245 1324
-4
E. coli
310,000
[2750]*
12
Chymotrypsin . . . . . . . . . . L-Lactate dehydrogenase.. 0VI4 isoenzyme) . . . . . . . . . Aspartate transcarbamylase
2 6 2 2 6 2 2 4 2.5 5.5
Reference Kendrew et gl (1958)36 Kendrew et al. (1960)37 Kendrew (1963) 37a Cullis et al (1962)38 Perutz (1969)3g Blake et al. (1962)40 Stanford et al (1962)41 Blake et al (1965)43 Lipscomb et al (1966)43 Lipscomb et al (1968)6 Kartha et al (1967)44 Wyckoff et al (1967)45 Wyckoff et al (1969)4e Matthews et al (1967)4~ Adams et al (1969)~8 Rossmann et al (1972)49 Warren et al (1973) a4
156
BLAIR
/~240-~~
,
._1 His 105
Hisbq i H2 i' HC--CO 2........ Arg+ 145 Zn IH N...
l
Olu " O ~ c ;'2 """" I
Fig. 1 - - The strzrct~tre of carboxypeptidase A , determi~ed by X - r a y analysis. (Reprod~(eed from the report by W . N. Lipscon~b '5°), with permissio~ of the a~thor and publishers.)
complex tl~at is relatively stable during the time required for X-ray diffraction measurements. Alternatively, a competitive inhibitor can be bound at the active site to form a stable, specific, enzyme--inhibitor complex; this locates the active site precisely and allows tentative identification of ammo acid sidechain groups involved in the catalytic reaction. The finding that crystals exhibit activity has encouraged the extrapolation of X-ray diffraction data to the catalytic behaviour of enzymes which normally operate in solution or in some special, defined cellular environment. In fact, ultimate elucidation of the mechanism of action of such an enzyme requires correlation of data from crystals with function data obtained from solutions. • Carboxypeptidase A specifically cleaves the carboxyl terminal residue from proteins; since it has been extensively studied with many different techniques, this enzyme provides an instructive example (Fig. 1). It is known '2~ that a single zinc atom is required for activity, and that this is located at the substrate binding site'S); thus, the position of the zinc atom indicates the active centre region (Fig. 1). Furthermore, crystals of carboxypeptidase are active and bind either substrate analogues ~ or very poorly reactive model substrates; these compounds appear in the zinc region of the electron-density map (4'5) The relationship between a bound substrate and the active-centre groups is highly specific. That for crystalline carboxypeptidase, depicted in Fig. 2, shows a hydrophobic 'pocket' into which the sidechain of the peptide carboxy-terminal residue fits, and binding groups for zinc (histidine-69, histidine196, and glutamic acid-72); zinc itself is present near the carbonyl group of the peptide bond to be cleaved. Chemical modification studies have sho~m that tyrosine and arginine residues are involved in peptidase activity (~'7~. In the X-ray-derived structure of the complex between glycyltyrosine and the A, *The active-site region of chymotrypsin, which is both an endopeptidase and esterase, has been delineated through the binding of substrate analogues; this enzyme contains no tightly bound essential metal.
..... H--O--Tyr 248
,' ':
Glu 210
Fig. '2 - - Relatio~ between a bo~e~ld s~bstrate a~d variotts active-centre grot{ps in carboxypeptidase A. (Re-drawn f r o m a representatio~ 's°~ by bY. N. Lipscomb a~d reprod~tced w i t h his permission.)
crystal form (which, elongated along the a axis, is used for X-ray diffraction studies), the side chains of tyrosine-248 and arginine-145 appear in the region of the substrate. Glutamic acid-270, also, appears sufficiently close to take part in the reaction'~L Catalysis by carboxypeptidase A has been discussed at length '7'~'. A possible mechanism based on the above specified amino-acid residues together with chemical and kinetic data is shown in Fig. 2. The main features are (1) proton transfer from tyrosine248 to the amide nitrogen, and (2) attack of a water molecule on the carbonyl carbon, which is polarised by its proximity to zinc; the attack by water could be facilitated by hydrogen-bond formation with glutamic acid-270. However, an alternative mechanism may be operative - - e.g., direct nucleophilic attack by a residue such as glutamic acid-270. The present data cannot be interpreted unequivocally. ~ ' This description of events at an active site is couched in terms of conventional physical organic chemistry. Can this approach fully explain the very high catalytic efficiency of enzymes in general? To do so, it is necessary to account for an enzymatic reaction velocity up to 10 ''~ times the rate of the corresponding uncatalysed reaction. The potential contributing factors of greatest current interest are (1) proximity, (2) orientation or 'orbital steering', (3) a non-aqueous micro-environment, and (4) strain. Although these terms have been common in biochemical parlance for some years, opinions concerning the relative significance of such factors are varied, assessments of their contributions being based mainly on calculations and extrapolations from simple model systems. In an oversimplified current view of this problem, the first factor to be considered is proximity. [This can be illustrated by the juxtaposition of zinc, glutamic acid-270, tyrosine-248, and the substrate, in carboxypeptidase (Fig. 2).] Certain calculations based on geometrical considerations can lead to the **Also, ester hydrolysis as catalysed by this same enzyme has not been considered here.
HOW AN ENZYME WORKS
157
h. IPIIJII'IPIIIIIII
+O>Q
+"
B. IIIj'~ll IV[
F-F-q
.... Q ( 3
+
-
II ~l?qlll+nlldl
t:, I'll I;AI Iv[ S
--
+
~"
/
Fig. ,~ - - Types of eonformational alterations that can occur i~+ a~ oligomerie protein with more than o~+e active site.
conclusion t h a t p r o x i m i t y itself is not of g r e a t importance. F o r example, if two r e a c t i n g molecules (A and B) a r e p r e s e n t in solution, each a t a c o n c e n t r a tion of 1 M, they m u s t f i n d e a c h - o t h e r a m o n g a prep o n d e r a n c e of w a t e r m o l e c u l e s ( c o n c e n t r a t i o n , 55 M) - - any A or B molecule is t y p i c a l l y s u r r o u n d e d by 12 w a t e r molecules. I f A is close to B (as at an enzyme active s i t e l , this c o n f e r s a r a t e a d v a n t a g e f o r reaction between the two. Most enzyme molecules, however, a r e p r e s e n t in the r e a c t i o n system at only 10-:' to 10 -* M, in which case the a d v a n t a g e a r i s i n g f r o m p r o x i m i t y is n e g l i g i b l e in r e l a t i o n to a c t u a l c a t a l y t i c r a t e s - - a conclusion t h a t has to be m o d i f i e d when several r e a c t i n g species a r e involved l e.g., subs t r a t e s and a c t i v e - s i t e r e s i d u e s ) , as a t an enzyme a c t i v e site'"' '"' W h a t e v e r i n t e r p r e t a t i o n is placed on p r o x i m i t y , some o r i e n t a t i o n s a r e u n p r o d u c t i v e : t h e r e a c t i o n r a t e should be s u b s t a n t i a l l y g r e a t e r when t h e enzyme g r o u p s and r e a c t i n g s u b s t r a t e molecules a r e v e r y precisely aligned in the active site'""~). T h i s is embodied in the concept of ' o r b i t a l s t e e r i n g ' advanced by Koshland '''~'''~. D a t a for i n t r a m o l e c u l a r r e a c t i o n s with model compounds s u g g e s t that, if the enzyme can m a i n t a i n o p t i m a l o r i e n t a t i o n , f o r some t y p e s of r e a c t i o n s the c r i t i c a l angle of the r e a c t i n g species m a y be small enough to give r i s e to r a t e a c c e l e r a t i o n of 10 ~ to 10L An i n t e r e s t i n g a p p r o a c h to a s s e s s m e n t of the relative c o n t r i b u t i o n s of p r o x i m i t y and o r i e n t a t i o n has been advanced by Jencks "~''s~. The above c o n s i d e r a tion of p r o x i m i t y f a i l s to t a k e into account t h e f a c t t h a t , even if r a n d o m m o v e m e n t s have displaced the i n t e r v e n i n g w a t e r molecules and have b r o u g h t A and B into close p r o x i m i t y , t h e y a r e still s u b j e c t to r a n dom m o v e m e n t : t h a t is, t h e y possess t r a n s l a t i o n a l and r o t a t i o n a l e n t r o p y . B i n d i n g to the enzyme ' f r e e z e s ' t h i s e n t r o p y and f a c i l i t a t e s collision and s u b s e q u e n t reaction. I n s e r t i o n of e n t r o p y f a c t o r s into the calculations has shown "~''+' t h a t p r o x i m i t y is a m a j o r d e t e r m i n a n t of the c a t a l y t i c e f f i c i e n c y of enzymes, a r e a s o n a b l e u p p e r l i m i t of its c o n t r i b u tion b e i n g a r a t e f a c t o r of 108. Obviously, t h i s po-
Fig. ~ - - St+'nctm'e of asparlate tra++scarbamylase. (Reprodnced f r o m the report by HZa~'+'e~ et al '~4', w i t h permissio++ o/ the a , t h o r a+~d pnblishers.)
t e n t i a l a d v a n t a g e is m u l t i p l i e d f o r r e a c t i o n s t h a t involve several s u b s t r a t e molecules and g r o u p s in t h e enzyme. The i m p l i c a t i o n s of special micro-envii-onments in the a c t i v e - c e n t r e region can be i l l u s t r a t e d by a g a i n c o n s i d e r i n g c a r b o x y p e p t i d a s e I F i g . 2), in which zinc bears a positive c h a r g e and the s u b s t r a t e c a r b o n y l oxygen is shown s u f f i c i e n t l y close to zinc to exclude w a t e r molecules. Thus, the e n v i r o n m e n t in t h i s locality would be e s s e n t i a l l y non-aqueous, so t h a t t h e e f f e c t i v e n e s s of c h a r g e p o l a r i s a t i o n of the carbonyl g r o u p by zinc would be much g r e a t e r t h a n if its e f f e c t s were t r a n s m i t t e d t h r o u g h i n t e r v e n i n g w a t e r molecule(s). Some enzymes induce s t e r i c d i s t o r t i o n in the subs t r a t e molecule t h a t f a v o u r s reaction. T h i s concept can be expressed in t e r m s of s u b s t r a t e - b i n d i n g e n e r g y used to d i s t o r t a p p r o p r i a t e bond angles so t h a t less e n e r g y is r e q u i r e d to reach the t r a n s i t i o n state, t h e r e b y i n c r e a s i n g the rate. B r u i c e ''7~ has p o i n t e d out t h a t such c o n f o r m a t i o n a l s t r a i n effects m a y be imp o r t a n t when the b i n d i n g e n e r g y of enzyme to s u b s t r a t e is s u f f i c i e n t l y g r e a t t h a t a p o r t i o n of t h i s e n e r g y may be expended in bond d i s t o r t i o n and y e t the b i n d i n g c o n s t a n t f o r s u b s t r a t e (K~) still r e m a i n r e a s o n a b l y small. Lysozyme, f o r which chemical and X - r a y c r y s t a l l o g r a p h y d a t a a r e in accord, has become a classic example of s t r a i n : in the p o l y s a c c h a r i d e s u b s t r a t e , the 6-membered hexose r i n g a d j a c e n t to the site of bond b r e a k a g e will not f i t p r o p e r l y in t h e active site unless d i s t o r t e d into h a l f - c h a i r c o n f o r m a tion - - and the t r a n s i t i o n - s t a t e i n t e r m e d i a t e carbon i u m ion a p p e a r s to have j u s t such a c o n f o r m a t i o n C~8~. Values f o r r a t e e n h a n c e m e n t s expected f o r each of these f a c t o r s will v a r y f r o m enzyme to enzyme. H o w - ' ever, t a k i n g all f o u r f a c t o r s into account, t h e t o t a l r a t e e n h a n c e m e n t p r e d i c t e d will reach t h e r a n g e o f c a t a l y t i c e f f i c i e n c y c h a r a c t e r i s t i c of enzymes. I t is emphasized t h a t , on t h i s basis, no special t h e o r i e s of c a t a l y s i s a r e r e q u i r e d to e x p l a i n the actions o f t h e s e large protein catalysts.
158
BLAIR
Modulation of substrate binding and catalysis at active s i t e s
Carboxypeptidase provides an example of conformational change relating to a single active site. X-ray diffraction studies of crystals have shown that the positions of certain residues vary by as much as 12 /~, depending upon whether the substrates are bound 'at. This supports Koshland's concept of induced fit, whereby groups in the active-centre region move to the correct catalytic alignment when the substrate is bound. However, these data apply to a particular crystal form of carboxypeptidase ( v i d e s u p r a ) , and Vallee and co-workers have obtained evidence of differences in the behaviour of this enzyme in solution and in other crystal forms ~'9). For purposes of this illustration the salient point is that the substrate induced a change in conformation by binding at the active site itself - - in an enzyme consisting of only one polypeptide chain and having only one active site. Enzymes consisting of more than one subunit, also, frequently undergo substrate-induced changes in conformation (analogous to those that occur in haemoglobin on 02 binding). Two theoretical treatments have proved particularly useful in interpreting the kinetics of such enzymes: the 'concerted model' proposed by Monod et al. '~°~ and Koshland's 'sequential model '~2'~. Fig. 3, which depicts three hypothetical enzymes each consisting of two subunits of identical primary structure with two active sites (one on each subunit), illustrates possible conformational changes at one a c t i v e site and how they may indirectly influence conformation at the other. Such conformational alterations, which are transmitted over large distances in the protein, are termed allosteric effects. However, transmission of a conformational change from one subunit to an adjacent subunit is not an obligatory concomitant of oligomeric structures: equation A in Fig. 3 shows two subunits that behave independently, the binding of a substrate molecule at one active site not affecting the binding of a second substrate molecule at the other active site. This does not preclude each subunit's undergoing a conformational change associated with substrate binding without influencing its neighbour (e.g., lactate dehydrogenase from bovine heart). The second allosteric case illustrated is that of positive co-operative interaction (Fig. 3, equations B i and it), in which binding of a substrate molecule at one site increases the affinity of the other active site for substrate. Conceptually, such a two-part change could take place in concert, provided that both subunits are in the same conformation whether it be of high or low affinity for substrate: the substrate merely alters the overall equilibrium in favour of the highaffinity state (equation Bi). Alternatively, according to the sequential model an alteration could be induced in the first subunit when the substrate binds, and thereby potentiate a conformational change in the adjacent subunit; because of greater resulting stabilisation of the overall oligomeric structure, the second subunit would have an increased tendency to alter conformation to the high-affinity state (equation B i t ) .
A large number of enzymes (e.g., threonine deaminase and phosphofructokinase) commonly show positive substrate-induced allosteric effects. At first it was not so well recognised that negative substrateinduced interactions, also, occur: equation C in Fig. 3 illustrates the induction of an altered conformation in the second subunit, characterised by reduced affinity for the second substrate molecule. This type of co-operative interaction was first delineated in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (2~,. Certain enzymes (e..a.. alkaline phosphatase from E. colt, and glyceraldehyde 3-phosphate dehydrogenase from yeast) display what has been termed "half-thesites reactivity ''~2'~'~', in which it appears that only half the supposedly identical active sites at a time can react with substrate. (The detailed mechanisms are relatively complex and will not be discussed here.) The kinetic consequences of positive allosteric interactions (Fig. 3, equation B) are well known. I f the initial reaction velocity is plotted as a function of substrate concentration, the resulting curve is sigmoid rather than hyperbolic. Negative co-operativity (equation C) results in an initial velocity v e r s u s substrate-concentration curve that appears to be hyperbolic but which can be distinguished by careful kinetic analysis. '~') With enzymes of more than two subunits, each with an active site, a mixture of positive and negative allosteric effects may be manifest on binding successive molecules of substrate. This condition can produce plateau regions in velocity v e r s u s substrate-concentration curves ''-7~. Allosteric models such as those depicted in Fig. 3 can be applied to substances whose structures differ radically from that of the substrate but which nevertheless interact with the enzyme to modify the reaction rate. Such 'modifiers' or 'effectors' bind at additional sites, distinct from the catalytic sites, on the subunits. Accordingly, the effects of modifiers on the active-site properties are also mediated by conformational changes that can be transmitted both within the subunit where the modifier binds and to neighbouring subunits. The kinetic consequences of allosteric changes involving modifiers may be complex, affecting either the substrate concentration required for half maximal velocity, or the maximal velocity, or both. The terminal product of a biosynthetic pathway often inhibits the first enzyme in that pathway, t h e r e b y - p r o v i d i n g feedback control of the total sequence - - which may involve several distinct enzymatically catalysed reactions. Aspartate transcarbamylase furnishes examples, in a single enzyme, of several types of these allosteric effects ~28). This member of the allosteric group has been more intensively examined than any other; the results of these studies were important in the early development of models to explain co-operative effects, particularly that devised by Monod et al. (2°). This concept embodied the restrictions that there are only two possible conformational states for any subunit and that all subunits must be in the same conformation; the derived rate equation was in satisfactory agTeement with the kinetic data available at that time (~g). Early studies with this enzyme suggested the presence of four catalytic and four regulatory
HOW AN ENZYME WORKS
s u b u n i t s p e r oligomer. Since then it has been shown t h a t a s p a r t a t e t r a n s c a r b a m y l a s e consists of 12 subunits - - six c a t a l y t i c , each w i t h an active site, and six r e g u l a t o r y s u b u n i t s each w i t h a b i n d i n g s i t e f o r t h e e f f e c t o r s (e.g., c y t i d i n e t r i p h o s p h a t e ) (3.'3t). T h e concerted model of Monod et al (2°) in its simple f o r m does not p r o v i d e an a d e q u a t e e x p l a n a t i o n of l a t e r k i n e t i c and b i n d i n g s t u d i e s w i t h t h i s e n z y m C 3"-). F o r example, the n a t i v e enzyme b i n d s t h r e e c y t i d i n e t r i p h o s p h a t e molecules r e a d i l y and an a d d i t i o n a l t h r e e with r e d u c e d a f f i n i t y (i.e., d e m o n s t r a t i n g n e g a t i v e co-operativity)'~3'. A r e c e n t l y p o s t u l a t e d s t r u c t u r e f o r a s p a r t a t e t r a n s c a r b a m y l a s e , based on X - r a y d i f f r a c tion s t u d i e s ' ~ ' ~ ' ( F i g . 4), shows the enzyme to be d i v i d e d into c a t a l y t i c and r e g u l a t o r y sections. The c a t a l y t i c p a r t s , in the f o r m of two t r i m e r s , a r e s e p a r a t e d by a middle section c o m p r i s i n g six r e g u l a t o r y s u b u n i t s . On the inside of t h e assembled molecules a r e t h e active sites exposed w i t h i n an aqueous cavity. T h e h y p o t h e s i s has been advanced t h a t s u b s t r a t e molecules reach the active s i t e s t h r o u g h channels (see f i g u r e ) , whose size can be v a r i e d b y b i n d i n g of both s u b s t r a t e and m o d i f i e r s . These subtle c h a n g e s in the t e r t i a r y s t r u c t u r e of enzymes ~ c h a n g e s t h a t can have such a s t r i k i n g e f f e c t on c a t a l y s i s - - a r e still i n c o m p l e t e l y u n d e r stood. T h e y will u n d o u b t e d l y be t h e s u b j e c t of much i n t e n s i v e i n v e s t i g a t i o n in t h e y e a r s ahead.
REFERENCES 1. Riordan, J. F. and Sokolovsky, M. (1971). Accounts Chem. Res. 4, 353 360. 2. Vallee, B. L. and Neurath, H. (1955). J. Biol. Chevy. 217, 253-261. 3. Coleman, J. E. and Vallee, B. L. (1964). Biochemistry 3, 1874-1879. 4. Steitz, T. A., Ludwig, M. L., Quicho, F. A. and Lipscomb, W. N. (1967). J. Biol. Chent. 242, 4662-4668. 5. Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H. and Coppola, J. C. (1968). Brookhaven Syrup. Biol. 21, 24-90. 6. Riordan, J. F. (1970). Federation Proe. 29, 462. 7. Vallee, B. L., Riordan, J. F., Auld, D. S. and Latt, S. A. (1970). Phil. Trans. Roy. Soc. London B257, 215-230. 8. Hartsuck, J. A. and Lipscomb, W. N. (1971). In The Enzymes, 3rd ed., ed. by P. D. Boyer. New York and London, Academic Press, vol. 3, 1-56. 9. Koshland, D. E., Jr. (1962). J. Theoret. Biol. 2, 75-86. 10. Koshland, D. E. and Neet, K. E. (1968). Ann. Rev. Bioehem. 37, 359-410. 11. Bruice, T. C. and Pandit, U. K. (1950). Proe. Natl. Acad. Sci. U.S.A., 46, 402-404. 12. Bruice, T. C. (1972). Nature (London) 237, 335-6. 13. Storm, D. R. and Koshland, D. E., Jr. (i970). Proe. Natl. Acad. Sci. U.S.A. 66, 445-452. 14. Dafforn, A. and Koshland, D. E. (1973). Bioehem. Biophys. Res. Commun. 52, 779-785. 15. Page, M. I. and Jencks, W. P. (1971). Proc. Natl. Acad. Sci. U.S.A. 68, 1678-1683. t 16. Jencks, W. P. (1973). P A A B A Revista 2, 235-254. 17. Bruice, T. C. (1970) in The Enzymes, 3rd ed., ed. by P. D. Boyer. New York and London, Academic Press vol. 3, 217-279. 18. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. and Rupley, J. A. (1972) in The Enzymes, 3rd ed., eel. by P. D. Boyer. New York and London, Academic Press vol. 7, 665-868. 19. Johansen, J. T. and Vallee, B. L. (1973). Proe. Natl. A ead. Sci. U.S.A. 70, 2006-2010.
159
20. Monod, J., W y m a n , J. and Changeux, J.-P. (1965). J. Mol. Biol. 12, 88-118. 21. Koshland, D. E., Jr., N~methy, G. and Filmer, D. (1966). Biochemistry 5, 365-385. 22. Conway, A. and Koshland, D. E., Jr., (1968). Biochemistry 7, 4011-4023. 23. Levitzki, A., Stalleup, W. B. and Koshland, D. E., Jr. (1971). Biochemistry 10, 3,371-3378. 24. Lazdunski, M., Petitclerc, C., Chappelet, D. and Lazdunski, F. (1971). Eur. J. Biochem. 20, 124-139. 25. Stallcup, W. B. and Koshland, D. E., Jr. (1973). J. Mol. Biol. 80, 77-91. 26. Koshland, D. E., Jr., (1970). In The Enzymes, 3rd ed., eu. oy r . D. x,ojer. New York and London, Academic Press, vol. 3, 341-396. 27. Teipel, J. and Koshland, D. E., Jr. (1969). Biochemistry 8, 4656-4663. 28. Gerhart, J. C. (1970). Curr. Top. Cell. Regul. 2, 275325. 29. Changeux, J.-P. and Rubin, M. (1968). Biochemistry 7, 553-561. 30. Weber, K. (1968). Nature (London) 218, 1116-1119. 31. Wiley, D. E. and Lipscomb, W. N. (1968). Nature (London) 218, 1119-1121. 32. Kerbiriou, D. and Herve, G. (1973). J. Mol. Biol. 78, 687-?02. 33. Wirdund, C. C. and Chamberlin, M. J. (1970). Bioehem. Biophys. Res. Commun. 40, 43-49. 34. Warren, S. G., Edwards, B. F. P., Evans, D. R., Wiley. D.C., and Lipscomb, W. N. (1973). Proe. Natl. Acad. Sci. U.S.A. 70, 1117-1121. 35. Evans, D. R., Warren, S. G., Edwards, B. F. P., MeMurray, C. H., Bethge, P. H., Wiley, D: C. and Lipscomb, W. N. (1973). Scienc6 179, 683-685. 36. Kenclrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H. and Phillips, D. C. (1958). Nature (London) 181, 662-666. 37. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. and Shore, V. C. (1960). Nature (London) 185, 422-427. 37. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. and Shore, V. C. (1960). Nature (London) 185, 422-427. 37a. Kendrew, J. C. (1963). Science 139, 1259-1266. 38. Cullis, A. F., Muirhead, H., Perutz, M. F., Rossmann, M. G. and North, A. C. T. (1962). Proc. Roy. Soe. A 265, 161-187. 39. Perutz, M. F. (1969). Proc. Roy. Soe. B. 173, 113-140. 40. Blake, C. C. F., Penn, R. H., North, A. C. T., Phillips, D. C., and Poljak, R. J. (1962). Nature (London) 196, 1173-1176. 41. Stanford, R. H., Jr., Marsh, R. E. and Corey, R. B. (1962). Nature (London) 196, 1176-1178. 42. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. and Sarma, V. R. (1965). Nature (London) 206, 757-761. 43. Lipscomb, W. N., Coppola, J. C., Hartsuck, J. A., Ludwig, M. L., Muirhead, H., Searl, J. and Steitz, T. A. (1966). J. Mol. Biol. 19, 423-441. 44. Kartha, G., Bello, J. and Harker, D. (1967). Nature (London) 213, 862-865. 45. Wyckoff, H. W., Hardman, K. D., Allewell, N. M., Inagami, T., Tsernoglou, D., Johnson, L. N. and Richards, F. M. (1967). J. Biol. Chem. 242, 3984-3988. 46. Wyckoff, H. W., Tsernoglou, D., Hanson, A. W., Knox, J. R., Lee, B. and Richards, F. M. (1970). J. Biol. Chem. 245, 305-328. 47. Mattbews, B. W., Sigler, P. B., Henderson, R. and Blow, D. M. (1967). Nature (London) 214, 652-656. 48. Adams, M. J., Haast, D. J., Jeffery, B. A., McPherson, A., Jr., Mermall, J. L., Rossmann, M. G., Schevitz, R. W. and Won.acott, A. J. (1969). J. Mol. Biol. 41, 159-188. 49. Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Lentz, P. J., Jr., McPherson, A., Jr., Schevitz, R. W. and Smiley, I. E. (1972). Cold Spring Harbor Syrup. Quant. Biol. 36, 179-191. 50. Lipscomb, W. N. (1970). Accounts Chem. Res. 3, 81-89.
How An Enzyme Works A. H. B L A I R Department
of B i o c h e m i s t r y , D a l h o u s i e U n i v e r s i t y .
Halifax, Nova
Scotia
B3H
4H7
well i l l u s t r a t e the development of this technique. Myoglobin, the f i r s t p r o t e i n for which the threed i m e n s i o n a l s t r u c t u r e was obtained (by Sir John K e n d r e w ) , is relatively small, c o n t a i n i n g only 153 residues. The work could be completed to a resolution of 6 A. before really f a s t computers were available to p e r f o r m the calculations, since only 400 reflections were required. By contrast, a resolution of 1.4 A (the limit of this technique) r e q u i r e d 25,000 reflections. Lactate dehydrogenase from dog-fish muscle cont a i n s a p p r o x i m a t e l y 1300 residues in four d i s t i n c t polypeptide c h a i n s ; solution to the 2.5 ~, level for the M., s u b u n i t r e q u i r e d calculations based on approximately 14,000 terms. Even at a resolution of 2.5 A, not all i n d i v i d u a l a m i n o acid residues can be i d e n t i f i e d with c e r t a i n t y f r o m X - r a y data alone, but require parallel use of the chemically d e t e r m i n e d amino acid sequence.
CLBIA, 9, (3) 155-159 (1976) Clin. Bioche~n.
Blair, A. H. D e p a r t m e n t of B i o c h e m i s t r y , Dalhousie [bHversit~./, H a l i f a x , N.S. B 3 H 4 H 7
HOW AN ENZYME WORKS The structure of enzyme active sites and the naturc of the catalytic process are reviewed. The impressive efficiency of these protein catalysts appears to stem from such factors as proximity and orientation of enzyme and substrate moieties, strain, and the occurrence of distinctive microenvironments within catalytic centres. Carboxypeptidase, lysozyme, and aspartate transcarbamylase, which have been extensively investigated by many techniques, serve tt, illustrate the application of these concepts. r
THIS BRIEF REVIEW ~:oncerns the s t r u c t u r e of enzyme active sites and the n a t u r e of the catalytic process. I t also outlines those factors associated with ligand b i n d i n g t h a t modulate events at an active site - - an aspect crucial to an u n d e r s t a n d i n g of how enzymes work in association with other cellular c o n s t i t u e n t s . To u n d e r s t a n d enzyme catalysis one m u s t relate s t r u c t u r e to f u n c t i o n . The sequence of amino-acid residues in an enzyme protein can be elucidated by wellestablished chemical m e t h o d s ; the t h r e e - d i m e n s i o n a l o r i e n t a t i o n and position of c o n s t i t u e n t residues can be d e t e r m i n e d by X - r a y d i f f r a c t i o n , which maps the e l e c t r o n - d e n s i t y d i s t r i b u t i o n . This t e c h n i q u e requires a protein t h a t will form defined c r y s t a l l i n e complexes with heavy atoms (e.g., H g ) . In recent years, a n u m b e r of enzymes and other p r o t e i n s have been mapped at high resolution (Table 1), and enzymes c o n s i s t i n g of multiple s u b u n i t s are now b e i n g investigated. Two proteins, namely myoglobin and lactate dehydrogenase,
The a m i n o acid residues t h a t are located at the active site (i.e., in a position to i n t e r a c t with the s u b s t r a t e d u r i n g catalysis) can be i d e n t i f i e d t h r o u g h selective chemical modification, by a n a l y s i n g the effect of m o d i f i c a t i o n on catalytic a c t i v i t y . " ' This approach has the a d v a n t a g e of dealing with the enzyme in solution, where conditions more closely a p p r o x i m a t e the physiological s t a t e ; however, lack of specificity in the reaction may complicate i n t e r p r e t a t i o n of results. Several approaches are possible for i d e n t i f y i n g the active-site region in an X - r a y - d e r i v e d s t r u c t u r e . F i r s t , if a t i g h t l y bound prosthetic group is involved in the reaction, this provides a m a r k e r t h a t will be a p p a r e n t in the X - r a y d i f f r a c t i o n p a t t e r n . These groups include metal ions (e.g., zinc in dehydrogenases). Second, and of potentially wider applicability, is the a c t i v i t y of some enzymes in t h e i r crystalline s t a t e : t h a t is, t h e i r s u b s t r a t e diffuses into the crystal to the active site, binds, and undergoes reaction. A very slowly r e a c t i n g s u b s t r a t e may form a
TABLE 1 X-RAY CRYSTALLOGRAPHYOF PROTEINS
Protein
Source
Mol wt
Number of residues
subunits
Resolution 6 2 1.4 5.5 2.8 6
Myoglobin . . . . . . . . . . . . . . .
Whale
17,800
153
--
Oxyhaemoglobin . . . . . . . . . .
Horse
64,500
574
4
Lysozyme . . . . . . . . . . . . . . . .
Hen egg
,
14,600
129
--
Carboxypeptidase . . . . . . . . .
Bovine pancreas
34,600
307
--
Ribonuclease. . . . . . . . . . . . . Ribonuclease S . . . . . . . . . . .
Bovine pancreas
13,683 13,700
124 124
---
Bovine pancreas Dog-fish muscle
25,000 150,000
245 1324
-4
E. coli
310,000
[2750]*
12
Chymotrypsin . . . . . . . . . . L-Lactate dehydrogenase.. 0VI4 isoenzyme) . . . . . . . . . Aspartate transcarbamylase
2 6 2 2 6 2 2 4 2.5 5.5
Reference Kendrew et gl (1958)36 Kendrew et al. (1960)37 Kendrew (1963) 37a Cullis et al (1962)38 Perutz (1969)3g Blake et al. (1962)40 Stanford et al (1962)41 Blake et al (1965)43 Lipscomb et al (1966)43 Lipscomb et al (1968)6 Kartha et al (1967)44 Wyckoff et al (1967)45 Wyckoff et al (1969)4e Matthews et al (1967)4~ Adams et al (1969)~8 Rossmann et al (1972)49 Warren et al (1973) a4
156
BLAIR
/~240-~~
,
._1 His 105
Hisbq i H2 i' HC--CO 2........ Arg+ 145 Zn IH N...
l
Olu " O ~ c ;'2 """" I
Fig. 1 - - The strzrct~tre of carboxypeptidase A , determi~ed by X - r a y analysis. (Reprod~(eed from the report by W . N. Lipscon~b '5°), with permissio~ of the a~thor and publishers.)
complex tl~at is relatively stable during the time required for X-ray diffraction measurements. Alternatively, a competitive inhibitor can be bound at the active site to form a stable, specific, enzyme--inhibitor complex; this locates the active site precisely and allows tentative identification of ammo acid sidechain groups involved in the catalytic reaction. The finding that crystals exhibit activity has encouraged the extrapolation of X-ray diffraction data to the catalytic behaviour of enzymes which normally operate in solution or in some special, defined cellular environment. In fact, ultimate elucidation of the mechanism of action of such an enzyme requires correlation of data from crystals with function data obtained from solutions. • Carboxypeptidase A specifically cleaves the carboxyl terminal residue from proteins; since it has been extensively studied with many different techniques, this enzyme provides an instructive example (Fig. 1). It is known '2~ that a single zinc atom is required for activity, and that this is located at the substrate binding site'S); thus, the position of the zinc atom indicates the active centre region (Fig. 1). Furthermore, crystals of carboxypeptidase are active and bind either substrate analogues ~ or very poorly reactive model substrates; these compounds appear in the zinc region of the electron-density map (4'5) The relationship between a bound substrate and the active-centre groups is highly specific. That for crystalline carboxypeptidase, depicted in Fig. 2, shows a hydrophobic 'pocket' into which the sidechain of the peptide carboxy-terminal residue fits, and binding groups for zinc (histidine-69, histidine196, and glutamic acid-72); zinc itself is present near the carbonyl group of the peptide bond to be cleaved. Chemical modification studies have sho~m that tyrosine and arginine residues are involved in peptidase activity (~'7~. In the X-ray-derived structure of the complex between glycyltyrosine and the A, *The active-site region of chymotrypsin, which is both an endopeptidase and esterase, has been delineated through the binding of substrate analogues; this enzyme contains no tightly bound essential metal.
..... H--O--Tyr 248
,' ':
Glu 210
Fig. '2 - - Relatio~ between a bo~e~ld s~bstrate a~d variotts active-centre grot{ps in carboxypeptidase A. (Re-drawn f r o m a representatio~ 's°~ by bY. N. Lipscomb a~d reprod~tced w i t h his permission.)
crystal form (which, elongated along the a axis, is used for X-ray diffraction studies), the side chains of tyrosine-248 and arginine-145 appear in the region of the substrate. Glutamic acid-270, also, appears sufficiently close to take part in the reaction'~L Catalysis by carboxypeptidase A has been discussed at length '7'~'. A possible mechanism based on the above specified amino-acid residues together with chemical and kinetic data is shown in Fig. 2. The main features are (1) proton transfer from tyrosine248 to the amide nitrogen, and (2) attack of a water molecule on the carbonyl carbon, which is polarised by its proximity to zinc; the attack by water could be facilitated by hydrogen-bond formation with glutamic acid-270. However, an alternative mechanism may be operative - - e.g., direct nucleophilic attack by a residue such as glutamic acid-270. The present data cannot be interpreted unequivocally. ~ ' This description of events at an active site is couched in terms of conventional physical organic chemistry. Can this approach fully explain the very high catalytic efficiency of enzymes in general? To do so, it is necessary to account for an enzymatic reaction velocity up to 10 ''~ times the rate of the corresponding uncatalysed reaction. The potential contributing factors of greatest current interest are (1) proximity, (2) orientation or 'orbital steering', (3) a non-aqueous micro-environment, and (4) strain. Although these terms have been common in biochemical parlance for some years, opinions concerning the relative significance of such factors are varied, assessments of their contributions being based mainly on calculations and extrapolations from simple model systems. In an oversimplified current view of this problem, the first factor to be considered is proximity. [This can be illustrated by the juxtaposition of zinc, glutamic acid-270, tyrosine-248, and the substrate, in carboxypeptidase (Fig. 2).] Certain calculations based on geometrical considerations can lead to the **Also, ester hydrolysis as catalysed by this same enzyme has not been considered here.
HOW AN ENZYME WORKS
157
h. IPIIJII'IPIIIIIII
+O>Q
+"
B. IIIj'~ll IV[
F-F-q
.... Q ( 3
+
-
II ~l?qlll+nlldl
t:, I'll I;AI Iv[ S
--
+
~"
/
Fig. ,~ - - Types of eonformational alterations that can occur i~+ a~ oligomerie protein with more than o~+e active site.
conclusion t h a t p r o x i m i t y itself is not of g r e a t importance. F o r example, if two r e a c t i n g molecules (A and B) a r e p r e s e n t in solution, each a t a c o n c e n t r a tion of 1 M, they m u s t f i n d e a c h - o t h e r a m o n g a prep o n d e r a n c e of w a t e r m o l e c u l e s ( c o n c e n t r a t i o n , 55 M) - - any A or B molecule is t y p i c a l l y s u r r o u n d e d by 12 w a t e r molecules. I f A is close to B (as at an enzyme active s i t e l , this c o n f e r s a r a t e a d v a n t a g e f o r reaction between the two. Most enzyme molecules, however, a r e p r e s e n t in the r e a c t i o n system at only 10-:' to 10 -* M, in which case the a d v a n t a g e a r i s i n g f r o m p r o x i m i t y is n e g l i g i b l e in r e l a t i o n to a c t u a l c a t a l y t i c r a t e s - - a conclusion t h a t has to be m o d i f i e d when several r e a c t i n g species a r e involved l e.g., subs t r a t e s and a c t i v e - s i t e r e s i d u e s ) , as a t an enzyme a c t i v e site'"' '"' W h a t e v e r i n t e r p r e t a t i o n is placed on p r o x i m i t y , some o r i e n t a t i o n s a r e u n p r o d u c t i v e : t h e r e a c t i o n r a t e should be s u b s t a n t i a l l y g r e a t e r when t h e enzyme g r o u p s and r e a c t i n g s u b s t r a t e molecules a r e v e r y precisely aligned in the active site'""~). T h i s is embodied in the concept of ' o r b i t a l s t e e r i n g ' advanced by Koshland '''~'''~. D a t a for i n t r a m o l e c u l a r r e a c t i o n s with model compounds s u g g e s t that, if the enzyme can m a i n t a i n o p t i m a l o r i e n t a t i o n , f o r some t y p e s of r e a c t i o n s the c r i t i c a l angle of the r e a c t i n g species m a y be small enough to give r i s e to r a t e a c c e l e r a t i o n of 10 ~ to 10L An i n t e r e s t i n g a p p r o a c h to a s s e s s m e n t of the relative c o n t r i b u t i o n s of p r o x i m i t y and o r i e n t a t i o n has been advanced by Jencks "~''s~. The above c o n s i d e r a tion of p r o x i m i t y f a i l s to t a k e into account t h e f a c t t h a t , even if r a n d o m m o v e m e n t s have displaced the i n t e r v e n i n g w a t e r molecules and have b r o u g h t A and B into close p r o x i m i t y , t h e y a r e still s u b j e c t to r a n dom m o v e m e n t : t h a t is, t h e y possess t r a n s l a t i o n a l and r o t a t i o n a l e n t r o p y . B i n d i n g to the enzyme ' f r e e z e s ' t h i s e n t r o p y and f a c i l i t a t e s collision and s u b s e q u e n t reaction. I n s e r t i o n of e n t r o p y f a c t o r s into the calculations has shown "~''+' t h a t p r o x i m i t y is a m a j o r d e t e r m i n a n t of the c a t a l y t i c e f f i c i e n c y of enzymes, a r e a s o n a b l e u p p e r l i m i t of its c o n t r i b u tion b e i n g a r a t e f a c t o r of 108. Obviously, t h i s po-
Fig. ~ - - St+'nctm'e of asparlate tra++scarbamylase. (Reprodnced f r o m the report by HZa~'+'e~ et al '~4', w i t h permissio++ o/ the a , t h o r a+~d pnblishers.)
t e n t i a l a d v a n t a g e is m u l t i p l i e d f o r r e a c t i o n s t h a t involve several s u b s t r a t e molecules and g r o u p s in t h e enzyme. The i m p l i c a t i o n s of special micro-envii-onments in the a c t i v e - c e n t r e region can be i l l u s t r a t e d by a g a i n c o n s i d e r i n g c a r b o x y p e p t i d a s e I F i g . 2), in which zinc bears a positive c h a r g e and the s u b s t r a t e c a r b o n y l oxygen is shown s u f f i c i e n t l y close to zinc to exclude w a t e r molecules. Thus, the e n v i r o n m e n t in t h i s locality would be e s s e n t i a l l y non-aqueous, so t h a t t h e e f f e c t i v e n e s s of c h a r g e p o l a r i s a t i o n of the carbonyl g r o u p by zinc would be much g r e a t e r t h a n if its e f f e c t s were t r a n s m i t t e d t h r o u g h i n t e r v e n i n g w a t e r molecule(s). Some enzymes induce s t e r i c d i s t o r t i o n in the subs t r a t e molecule t h a t f a v o u r s reaction. T h i s concept can be expressed in t e r m s of s u b s t r a t e - b i n d i n g e n e r g y used to d i s t o r t a p p r o p r i a t e bond angles so t h a t less e n e r g y is r e q u i r e d to reach the t r a n s i t i o n state, t h e r e b y i n c r e a s i n g the rate. B r u i c e ''7~ has p o i n t e d out t h a t such c o n f o r m a t i o n a l s t r a i n effects m a y be imp o r t a n t when the b i n d i n g e n e r g y of enzyme to s u b s t r a t e is s u f f i c i e n t l y g r e a t t h a t a p o r t i o n of t h i s e n e r g y may be expended in bond d i s t o r t i o n and y e t the b i n d i n g c o n s t a n t f o r s u b s t r a t e (K~) still r e m a i n r e a s o n a b l y small. Lysozyme, f o r which chemical and X - r a y c r y s t a l l o g r a p h y d a t a a r e in accord, has become a classic example of s t r a i n : in the p o l y s a c c h a r i d e s u b s t r a t e , the 6-membered hexose r i n g a d j a c e n t to the site of bond b r e a k a g e will not f i t p r o p e r l y in t h e active site unless d i s t o r t e d into h a l f - c h a i r c o n f o r m a tion - - and the t r a n s i t i o n - s t a t e i n t e r m e d i a t e carbon i u m ion a p p e a r s to have j u s t such a c o n f o r m a t i o n C~8~. Values f o r r a t e e n h a n c e m e n t s expected f o r each of these f a c t o r s will v a r y f r o m enzyme to enzyme. H o w - ' ever, t a k i n g all f o u r f a c t o r s into account, t h e t o t a l r a t e e n h a n c e m e n t p r e d i c t e d will reach t h e r a n g e o f c a t a l y t i c e f f i c i e n c y c h a r a c t e r i s t i c of enzymes. I t is emphasized t h a t , on t h i s basis, no special t h e o r i e s of c a t a l y s i s a r e r e q u i r e d to e x p l a i n the actions o f t h e s e large protein catalysts.
158
BLAIR
Modulation of substrate binding and catalysis at active s i t e s
Carboxypeptidase provides an example of conformational change relating to a single active site. X-ray diffraction studies of crystals have shown that the positions of certain residues vary by as much as 12 /~, depending upon whether the substrates are bound 'at. This supports Koshland's concept of induced fit, whereby groups in the active-centre region move to the correct catalytic alignment when the substrate is bound. However, these data apply to a particular crystal form of carboxypeptidase ( v i d e s u p r a ) , and Vallee and co-workers have obtained evidence of differences in the behaviour of this enzyme in solution and in other crystal forms ~'9). For purposes of this illustration the salient point is that the substrate induced a change in conformation by binding at the active site itself - - in an enzyme consisting of only one polypeptide chain and having only one active site. Enzymes consisting of more than one subunit, also, frequently undergo substrate-induced changes in conformation (analogous to those that occur in haemoglobin on 02 binding). Two theoretical treatments have proved particularly useful in interpreting the kinetics of such enzymes: the 'concerted model' proposed by Monod et al. '~°~ and Koshland's 'sequential model '~2'~. Fig. 3, which depicts three hypothetical enzymes each consisting of two subunits of identical primary structure with two active sites (one on each subunit), illustrates possible conformational changes at one a c t i v e site and how they may indirectly influence conformation at the other. Such conformational alterations, which are transmitted over large distances in the protein, are termed allosteric effects. However, transmission of a conformational change from one subunit to an adjacent subunit is not an obligatory concomitant of oligomeric structures: equation A in Fig. 3 shows two subunits that behave independently, the binding of a substrate molecule at one active site not affecting the binding of a second substrate molecule at the other active site. This does not preclude each subunit's undergoing a conformational change associated with substrate binding without influencing its neighbour (e.g., lactate dehydrogenase from bovine heart). The second allosteric case illustrated is that of positive co-operative interaction (Fig. 3, equations B i and it), in which binding of a substrate molecule at one site increases the affinity of the other active site for substrate. Conceptually, such a two-part change could take place in concert, provided that both subunits are in the same conformation whether it be of high or low affinity for substrate: the substrate merely alters the overall equilibrium in favour of the highaffinity state (equation Bi). Alternatively, according to the sequential model an alteration could be induced in the first subunit when the substrate binds, and thereby potentiate a conformational change in the adjacent subunit; because of greater resulting stabilisation of the overall oligomeric structure, the second subunit would have an increased tendency to alter conformation to the high-affinity state (equation B i t ) .
A large number of enzymes (e.g., threonine deaminase and phosphofructokinase) commonly show positive substrate-induced allosteric effects. At first it was not so well recognised that negative substrateinduced interactions, also, occur: equation C in Fig. 3 illustrates the induction of an altered conformation in the second subunit, characterised by reduced affinity for the second substrate molecule. This type of co-operative interaction was first delineated in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (2~,. Certain enzymes (e..a.. alkaline phosphatase from E. colt, and glyceraldehyde 3-phosphate dehydrogenase from yeast) display what has been termed "half-thesites reactivity ''~2'~'~', in which it appears that only half the supposedly identical active sites at a time can react with substrate. (The detailed mechanisms are relatively complex and will not be discussed here.) The kinetic consequences of positive allosteric interactions (Fig. 3, equation B) are well known. I f the initial reaction velocity is plotted as a function of substrate concentration, the resulting curve is sigmoid rather than hyperbolic. Negative co-operativity (equation C) results in an initial velocity v e r s u s substrate-concentration curve that appears to be hyperbolic but which can be distinguished by careful kinetic analysis. '~') With enzymes of more than two subunits, each with an active site, a mixture of positive and negative allosteric effects may be manifest on binding successive molecules of substrate. This condition can produce plateau regions in velocity v e r s u s substrate-concentration curves ''-7~. Allosteric models such as those depicted in Fig. 3 can be applied to substances whose structures differ radically from that of the substrate but which nevertheless interact with the enzyme to modify the reaction rate. Such 'modifiers' or 'effectors' bind at additional sites, distinct from the catalytic sites, on the subunits. Accordingly, the effects of modifiers on the active-site properties are also mediated by conformational changes that can be transmitted both within the subunit where the modifier binds and to neighbouring subunits. The kinetic consequences of allosteric changes involving modifiers may be complex, affecting either the substrate concentration required for half maximal velocity, or the maximal velocity, or both. The terminal product of a biosynthetic pathway often inhibits the first enzyme in that pathway, t h e r e b y - p r o v i d i n g feedback control of the total sequence - - which may involve several distinct enzymatically catalysed reactions. Aspartate transcarbamylase furnishes examples, in a single enzyme, of several types of these allosteric effects ~28). This member of the allosteric group has been more intensively examined than any other; the results of these studies were important in the early development of models to explain co-operative effects, particularly that devised by Monod et al. (2°). This concept embodied the restrictions that there are only two possible conformational states for any subunit and that all subunits must be in the same conformation; the derived rate equation was in satisfactory agTeement with the kinetic data available at that time (~g). Early studies with this enzyme suggested the presence of four catalytic and four regulatory
HOW AN ENZYME WORKS
s u b u n i t s p e r oligomer. Since then it has been shown t h a t a s p a r t a t e t r a n s c a r b a m y l a s e consists of 12 subunits - - six c a t a l y t i c , each w i t h an active site, and six r e g u l a t o r y s u b u n i t s each w i t h a b i n d i n g s i t e f o r t h e e f f e c t o r s (e.g., c y t i d i n e t r i p h o s p h a t e ) (3.'3t). T h e concerted model of Monod et al (2°) in its simple f o r m does not p r o v i d e an a d e q u a t e e x p l a n a t i o n of l a t e r k i n e t i c and b i n d i n g s t u d i e s w i t h t h i s e n z y m C 3"-). F o r example, the n a t i v e enzyme b i n d s t h r e e c y t i d i n e t r i p h o s p h a t e molecules r e a d i l y and an a d d i t i o n a l t h r e e with r e d u c e d a f f i n i t y (i.e., d e m o n s t r a t i n g n e g a t i v e co-operativity)'~3'. A r e c e n t l y p o s t u l a t e d s t r u c t u r e f o r a s p a r t a t e t r a n s c a r b a m y l a s e , based on X - r a y d i f f r a c tion s t u d i e s ' ~ ' ~ ' ( F i g . 4), shows the enzyme to be d i v i d e d into c a t a l y t i c and r e g u l a t o r y sections. The c a t a l y t i c p a r t s , in the f o r m of two t r i m e r s , a r e s e p a r a t e d by a middle section c o m p r i s i n g six r e g u l a t o r y s u b u n i t s . On the inside of t h e assembled molecules a r e t h e active sites exposed w i t h i n an aqueous cavity. T h e h y p o t h e s i s has been advanced t h a t s u b s t r a t e molecules reach the active s i t e s t h r o u g h channels (see f i g u r e ) , whose size can be v a r i e d b y b i n d i n g of both s u b s t r a t e and m o d i f i e r s . These subtle c h a n g e s in the t e r t i a r y s t r u c t u r e of enzymes ~ c h a n g e s t h a t can have such a s t r i k i n g e f f e c t on c a t a l y s i s - - a r e still i n c o m p l e t e l y u n d e r stood. T h e y will u n d o u b t e d l y be t h e s u b j e c t of much i n t e n s i v e i n v e s t i g a t i o n in t h e y e a r s ahead.
REFERENCES 1. Riordan, J. F. and Sokolovsky, M. (1971). Accounts Chem. Res. 4, 353 360. 2. Vallee, B. L. and Neurath, H. (1955). J. Biol. Chevy. 217, 253-261. 3. Coleman, J. E. and Vallee, B. L. (1964). Biochemistry 3, 1874-1879. 4. Steitz, T. A., Ludwig, M. L., Quicho, F. A. and Lipscomb, W. N. (1967). J. Biol. Chent. 242, 4662-4668. 5. Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H. and Coppola, J. C. (1968). Brookhaven Syrup. Biol. 21, 24-90. 6. Riordan, J. F. (1970). Federation Proe. 29, 462. 7. Vallee, B. L., Riordan, J. F., Auld, D. S. and Latt, S. A. (1970). Phil. Trans. Roy. Soc. London B257, 215-230. 8. Hartsuck, J. A. and Lipscomb, W. N. (1971). In The Enzymes, 3rd ed., ed. by P. D. Boyer. New York and London, Academic Press, vol. 3, 1-56. 9. Koshland, D. E., Jr. (1962). J. Theoret. Biol. 2, 75-86. 10. Koshland, D. E. and Neet, K. E. (1968). Ann. Rev. Bioehem. 37, 359-410. 11. Bruice, T. C. and Pandit, U. K. (1950). Proe. Natl. Acad. Sci. U.S.A., 46, 402-404. 12. Bruice, T. C. (1972). Nature (London) 237, 335-6. 13. Storm, D. R. and Koshland, D. E., Jr. (i970). Proe. Natl. Acad. Sci. U.S.A. 66, 445-452. 14. Dafforn, A. and Koshland, D. E. (1973). Bioehem. Biophys. Res. Commun. 52, 779-785. 15. Page, M. I. and Jencks, W. P. (1971). Proc. Natl. Acad. Sci. U.S.A. 68, 1678-1683. t 16. Jencks, W. P. (1973). P A A B A Revista 2, 235-254. 17. Bruice, T. C. (1970) in The Enzymes, 3rd ed., ed. by P. D. Boyer. New York and London, Academic Press vol. 3, 217-279. 18. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. and Rupley, J. A. (1972) in The Enzymes, 3rd ed., eel. by P. D. Boyer. New York and London, Academic Press vol. 7, 665-868. 19. Johansen, J. T. and Vallee, B. L. (1973). Proe. Natl. A ead. Sci. U.S.A. 70, 2006-2010.
159
20. Monod, J., W y m a n , J. and Changeux, J.-P. (1965). J. Mol. Biol. 12, 88-118. 21. Koshland, D. E., Jr., N~methy, G. and Filmer, D. (1966). Biochemistry 5, 365-385. 22. Conway, A. and Koshland, D. E., Jr., (1968). Biochemistry 7, 4011-4023. 23. Levitzki, A., Stalleup, W. B. and Koshland, D. E., Jr. (1971). Biochemistry 10, 3,371-3378. 24. Lazdunski, M., Petitclerc, C., Chappelet, D. and Lazdunski, F. (1971). Eur. J. Biochem. 20, 124-139. 25. Stallcup, W. B. and Koshland, D. E., Jr. (1973). J. Mol. Biol. 80, 77-91. 26. Koshland, D. E., Jr., (1970). In The Enzymes, 3rd ed., eu. oy r . D. x,ojer. New York and London, Academic Press, vol. 3, 341-396. 27. Teipel, J. and Koshland, D. E., Jr. (1969). Biochemistry 8, 4656-4663. 28. Gerhart, J. C. (1970). Curr. Top. Cell. Regul. 2, 275325. 29. Changeux, J.-P. and Rubin, M. (1968). Biochemistry 7, 553-561. 30. Weber, K. (1968). Nature (London) 218, 1116-1119. 31. Wiley, D. E. and Lipscomb, W. N. (1968). Nature (London) 218, 1119-1121. 32. Kerbiriou, D. and Herve, G. (1973). J. Mol. Biol. 78, 687-?02. 33. Wirdund, C. C. and Chamberlin, M. J. (1970). Bioehem. Biophys. Res. Commun. 40, 43-49. 34. Warren, S. G., Edwards, B. F. P., Evans, D. R., Wiley. D.C., and Lipscomb, W. N. (1973). Proe. Natl. Acad. Sci. U.S.A. 70, 1117-1121. 35. Evans, D. R., Warren, S. G., Edwards, B. F. P., MeMurray, C. H., Bethge, P. H., Wiley, D: C. and Lipscomb, W. N. (1973). Scienc6 179, 683-685. 36. Kenclrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H. and Phillips, D. C. (1958). Nature (London) 181, 662-666. 37. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. and Shore, V. C. (1960). Nature (London) 185, 422-427. 37. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. and Shore, V. C. (1960). Nature (London) 185, 422-427. 37a. Kendrew, J. C. (1963). Science 139, 1259-1266. 38. Cullis, A. F., Muirhead, H., Perutz, M. F., Rossmann, M. G. and North, A. C. T. (1962). Proc. Roy. Soe. A 265, 161-187. 39. Perutz, M. F. (1969). Proc. Roy. Soe. B. 173, 113-140. 40. Blake, C. C. F., Penn, R. H., North, A. C. T., Phillips, D. C., and Poljak, R. J. (1962). Nature (London) 196, 1173-1176. 41. Stanford, R. H., Jr., Marsh, R. E. and Corey, R. B. (1962). Nature (London) 196, 1176-1178. 42. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. and Sarma, V. R. (1965). Nature (London) 206, 757-761. 43. Lipscomb, W. N., Coppola, J. C., Hartsuck, J. A., Ludwig, M. L., Muirhead, H., Searl, J. and Steitz, T. A. (1966). J. Mol. Biol. 19, 423-441. 44. Kartha, G., Bello, J. and Harker, D. (1967). Nature (London) 213, 862-865. 45. Wyckoff, H. W., Hardman, K. D., Allewell, N. M., Inagami, T., Tsernoglou, D., Johnson, L. N. and Richards, F. M. (1967). J. Biol. Chem. 242, 3984-3988. 46. Wyckoff, H. W., Tsernoglou, D., Hanson, A. W., Knox, J. R., Lee, B. and Richards, F. M. (1970). J. Biol. Chem. 245, 305-328. 47. Mattbews, B. W., Sigler, P. B., Henderson, R. and Blow, D. M. (1967). Nature (London) 214, 652-656. 48. Adams, M. J., Haast, D. J., Jeffery, B. A., McPherson, A., Jr., Mermall, J. L., Rossmann, M. G., Schevitz, R. W. and Won.acott, A. J. (1969). J. Mol. Biol. 41, 159-188. 49. Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Lentz, P. J., Jr., McPherson, A., Jr., Schevitz, R. W. and Smiley, I. E. (1972). Cold Spring Harbor Syrup. Quant. Biol. 36, 179-191. 50. Lipscomb, W. N. (1970). Accounts Chem. Res. 3, 81-89.
