Protein Science (1992), I , 582-589. Cambridge University Press. Printed in the USA.

Copyright 0 1992 The Protein Society

Specificity determinants of acylaminoacyl-peptide hydrolase

RADHA G . KRISHNA AND FINN WOLD Department of Biochemistry and Molecular Biology, University of Texas Medical School, P.O. Box 20708, Houston, Texas 77225 (RECEIVEDNovember 8, 1991; REVISEDMANUSCRIPT RECEIVED December 24, 1991)

Abstract In an attempt to explore how specific features of the substrate’s primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19. l), a number of acetylated peptides containingspecific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effectof the length of the peptide was also investigated in a less systematic manner. The substrateswere either prepared by direct acetNylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as hydroxysuccinimide esters, or by isolation of theN-terminal peptides from naturally occurringacetylated proteins. It was found that D-Ala on either side of the bond to becleaved (positions 1 and 2 ) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2070 if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2 , 3 , 4 , 5 , and 6 gave strong reduction in hydrolaseactivity varying with the charge’s distance from theN-terminus from 0 to 15-20% of therates obtained with the reference peptides without positive charges. Deprotonation of His at high pH generatedexcellent substrates, and removal of the positive charges of Lys by acetylation or, even better, succinylation also gave improved substrate quality, demonstrating that the positive charges are responsible for the inhibition. Long peptides (10-29 residues) were generally found to be poor substrates, especially when they contained positive charges and Pro. The better long peptide substrates d o not have these residues, but contain negative charges instead. A survey of the N-terminal sequences of more than 100 acetylated proteins revealed that about 95% of them have Pro and/or positively charged amino acids among the first10 residues, suggesting that these residues may be natural inhibitors of hydrolase action in vivo. In addition to the specific and large effect of the residues described above on substrate quality, it also appears that there is a general effect of the overall sequence of each peptide, and that thespecific effects of individual residues are modulated significantly by the environment (context) in which they are expressed.

Keywords: acetylated N-termini; acylaminoacyl-peptide hydrolase; N-terminal processing; ification; primary sequence specificity determinants

A large number of chemical modifications of proteins take place in living cells both during co- and posttranslational steps of protein biosynthesis, during signal transduction and the regulation of biological activity, during

posttranslational mod-

transport, and during proteolytic destruction. Most of these reactions are characterized by an extremely high degree of specificity; eventhe least specific reactions involve only a few amino acid residues, and in some cases only a single residue is modified or a single peptide bond is cleaved. The different reactions take place in many difReprintrequeststo:Dr.FinnWold,Department of Biochemistry and ferent compartments and at different stages of protein Molecular Biology, Universityof Texas Medical School,P.O. Box 20708, synthesis and transport, and to understand all the speciHouston, Texas 77225. I This enzymatic activity, N-acylaminoacyl-peptide hydrolase, has ficity determinants directing the modifications, it is necalso been referred to as acetylaminoacyl-peptide hydrolase, acylpeptide essary to consider all levels of structural organization in hydrolase, acyl-peptide hydrolase, N-acetylalanine aminopeptidase, and acylamino acid-releasing enzyme in the literature. It is likely, but by no proteins*For short nascent chains the dominant specificmeans proven, that these are all the same or closely related enzymes. ity determinants are derived primarily from the primary 582

583

Specifcity of acylaminoacyl-peptide hydrolase sequence. For longer nascent chains and completed polymers, however, both secondary, tertiary, and quaternary structural features, often perhaps only transient ones, must also affect the interaction of the protein with the processing enzymes and receptors. The N-terminal processing steps in protein synthesis appear to be mostly early events in protein synthesis (Arfin & Bradshaw, 1988) and presumably involve primary structure as the major source of unique specificity signalsfor the processing enzymes. In anattempt to assess the kind of specificity signalsthat may be involvedat this level of structure, we have expanded our previous studies (Radhakrishna & Wold, 1989) of the action of the enzyme N-acylaminoacyl-peptide hydrolase(EC 3.4.19.1) on anumber of peptides of different sequence and length. Based on the past work on a number of preparations of this enzyme in several laboratories,’ it is known that the enzyme is active in catalyzing the cleavage of Ac-Ala, Ac-Thr, Ac-Met, Ac-Gly, and Ac-Ser from the N-terminal end of relatively short peptides; it also removes some other Ac-amino acids (e.g., Cys, Val), but with much lower activity. No intact acetylated protein has yet been found that is a substrate for the enzyme. Peptides up to 12 residues long have been found to be good substrates, whereas others,

even shorter ones, are essentially resistant to the hydrolase, and it is clear that a number of primary structural features must beaccounted for in the analysis of the specificity of the hydrolase. In this paper we explore some of these features and present the current status of the specificity requirements of the hydrolase. Results The apparent rate constantsobserved for the hydrolasecatalyzed hydrolysis of a large number of different acetylated peptides are listed in Tables 1 and 2. The tables have been organized to emphasize the negative effect of certain amino acid residues in certain positions of the substrates and also to reflect the general decrease in rate as the peptides grow longer. To evaluate the new data, it is important to summarize earlier observations (Tsunasawa et al., 1975; Gade & Brown, 1978; Jones & Manning, 1985; Kobayashi & Smith, 1987; Radhakrishna & Wold, 1989) regarding the specificity of this enzyme. Briefly, it has been shown that the enzyme requires one of five N-terminal acetylamino acidsfor rapid hydrolysis: Thr, Ala, Met, Ser, Gly; from comparison of peptides differing only in the N-terminal amino acid, it appears

Table 1. Specificity of acylaminoacyl-peptide hydrolase: the effect of proline, D-amino acids, and positive charges on substrate quality a substrate A. Proline

Reference

10’ x k (min”)

Ac-AAA

6,950

Substrate 2,438

Ac-GGG Ac-AAAA Ac-AAAAA Ac-AAAAAA

1,453 4,896 3,871 3,792 2,841

Ac-APA Ac-AAP Ac-GGP Ac-AAPA Ac-AAPAA Ac-AAAPA Ac-AGAAPA

10’ x k (min”)

14 65 72 98 2,856

B. D-Amino acidsb

C. Positive charges Histidine

Arginine

LysineC

Ac-AA Ac-AAA Ac-AAAA Ac-AAAAA

965 6,950 4,896 3,871

Ac-AA Ac-AA A Ac-AGAA Ac-AGAAA

4,825 3,885

Ac-AHA Ac-AH+A Ac-AAHA

8,470 6,060 5,352

Ac-AAH+A

< 10 < 10

Ac-AAAAAA

2,841

Ac-AAARG Ac-AGAARG Ac-AAGAARG

Ac-AAGGDASGE Ac-AAQJ Ac-AAQU

3,438 5,280 6,849

Ac-AAQK

Ac-AAFG Ac-AARG

0 95

424 450

598 563 1,029

a The columns on the left in general contain the reference (“good”) substrates of the same or similar length and composition as the experimental ones, which are listed in the right-hand column. D-Ala is indicated by boldface A in the structures. The letters J and U are used to designate Nf-acetyl-Lys and Ne-succinyl-Lys, respectively.

5 84

R.G. Krishna and F. Wold Table 2. The specificity of acylaminoacyl-peptide hydrolase: the effect of different substituent amino acids and peptide length on substrate quality“ Reference substrate Ac-AAGGDASGE Ac-AAQKRPS Ac-MAGGDASGE Ac-AAQJRPS Ac-GAGGDASGE Ac-MDETGDTALVA

IO5 x k (min-I)

3,438 1,812 212 2,068

Substrate

lo5 x k (min-I) 200 561

Ac-AAQURPS Ac-AAQKRPSQRSKY Ac-AAQJRPSQRSJY Ac-AAQURPSQRSUY Ac-AAQKRPSQRSKYLASASTX Ac-AAQJRPSQRSJYLASASTX Ac-AAQURPSQRSUYLASASTX

AC-AGIVEQCCASVCSLYQLENYCN~ Ac-AFVNQHLCCSHLVEALYLVCGERGFFYTP‘

695 19 58 83 12 33 39 83 18

a The column to the left represents fairly long (9 and 1 1 residues) peptides that are “good” substrates (Radhakrishna & Wold, 1989). The one on the right shows the data for three of the four myelin basic protein N-terminal peptides with and without the Lys modifications (J and U are used as symbols for Ne-acetyl-Lys and Ne-succinyl-Lys, respectively, and X signifies a mixture of homoserine and homoserine lactone) and the derivatives of oxidized insulin chains A and B (C signifies cysteic acid). Only (Ac-A)-InsA is shown. The rate constants determined for the other derivatives are Ac-InsA, 11; (Ac-M)-InsA, 54; and (Ac-G)-InsA, 10. Only (Ac-A)-desKA-InsB is shown. The rate constants determined for the other derivatives are Ac-desKA-InsB, 0; (Ac-M)desKA-InsB, 11; and (Ac-G)-desKA-InsB, 4.

that Ac-Thr is cleaved 5-6 times faster than Ac-Gly, and the remaining three fall between these values in the order given. Acetylated N-terminals involving Cys, Val, Phe, Tyr, and Asp are also cleaved from short acetylated peptides, but at a rate less than 1Yo of that with the optimal substrates. It was also shown that Pro or Arg in the second position of the peptide would reduce the hydrolysis rate about 100-fold. Although it appears that the enzyme is optimally active on short peptides containing 3-4 residues, peptides with 9 and 11 residues were found to be excellent substrates for the hydrolase, having rate constants 20-50% of those for the corresponding tripeptides; it may be significant that these longer peptidescontained either 2 or 3 negatively charged (Asp and/or Glu) residues. The present study was undertaken to further explore the observed effect of Arg and Pro in the second position. In the case of Pro as the second residue in the peptide, the bond to be broken involves an imide instead of the normal amide bond, and as it is well established that most proteases are unable to cleave the imide bonds, it is perhaps not surprising that the hydrolase also shows very low activity with these peptides. However, as is shown in Table 1, there is also a very significant effectof Pro when it is located in the third position. For the tripeptide Ac-AAP this effect is not very large, but if additional residues are present (Ac-AAPAand Ac-AAPAA) the hydrolysis rate is 40-70 times lower than for the reference peptides. The peptide Ac-GGP is different from the Ac-AAP peptide in being a poor substratein the absence of additional C-terminal residues. Even allowing

for the fact that Ac-Gly-peptides have the lowest rate of hydrolysis of the five groups of primary substrates, the rate decrease for this peptide is large enough to suggest that some unique property may have to be considered. Pro in the fourth or fifth position does not have any significant effect with the present peptides, but the negative influence of Pro in the third position will have to be accounted for in any model aimed at understanding the specificity of the hydrolase. The effect of D-alanine (Table 1, B) reflects a more straightforward influence of the stereochemistry of the two amino acids involved in the peptide bond to be broken. D-Ala in position 3 or 4 appears to be completely equivalent to the natural enantiomer. The effect of positive charges in the substrate on the rate of the reaction is illustrated in Table 1, C. We reported earlier that Arg in the second position was one of the strongest negative effectors of the rate of the hydrolase reaction(Radhakrishna & Wold, 1989, 1990), and the following substrates were included inthe present study to establish whetherthe effect was specifically due to Arg or represented a general effect of positive charges. Because His can be titrated to be either positively charged or neutral in the pH range in which the enzyme is active, His-containing peptides were prepared and subjected to hydrolase-catalyzed hydrolysis at different pH values, using Ac-AAA and Ac-AAAA as standard nonionizing peptides to normalize the enzyme’s activity. The results are given in Figure 1 and demonstrate that although the peptides with the neutral deprotonated His in either the second or the third position are excellent substrates for

585

Specifcity of acylaminoacyl-peptide hydrolase

Table 3. Effect of Arg and Pro on the kinetic parameters of acylaminoacyl-peptide hydrolase

>

f? 40 U

4.5 5.0 5.5

6.0 6.5 7.0 7.5 8.0 8.5

9.0

PH Fig. 1. The effect of pH on the rate of hydrolysis of His-containing acetylpeptides. The rate of hydrolysis of Ac-AHA, Ac-AAHA, Ac-AAA, and Ac-AAAA was determined under standard conditions of enzyme concentration and temperature, using 50 mM NaOAc buffer, pH 5.0 and pH 5.5, 50 mM (Na)P04 buffer, pH 6.0 and pH 6.9, and 50 mM Tris-HC1 buffer, pH 8.7; all buffers containing1 mM EDTA and 2 mM MgCI2. (The pH6.9 buffer is the standard assay buffer.) To correct for the effect of pH on the hydrolaseitself, the rates at different pH for the two nonionizing substratesAc-AAA and Ac-AAAA were first normalized to the pH 6.9 rate as 100% activity (. . . . .). The appropriate normalization factors were then appliedto the observed hydrolysis rates for Ac-AHA ( 0 ) and Ac-AAHA (o),and the rate vs. pH curves were plotted using the corrected pH8.7 rate as the 100% activity for thetwo Hiscontaining substrates. The apparent pK values for Ac-AHA and Ac-AAHA are 6.72 and 6.79, respectively.

the hydrolase, the protonated, positively charged forms of the same substrates are essentially inert inthe reaction. The activity of the enzyme at the lower pH values is too low to permit an accurate assessment of the activity with the protonated substrates. The rate constants obtained with the His-containing substrates are reported in Table 1 along with those determined for a series of Arg-and Lyscontaining peptides. The Arg peptides confirm the inhibitory effect of the positive charges, giving a 10-fold decrease in the rate when Arg is in the third position to a 4-5-fold decrease when it is in the sixth position. Only one peptide containing Lys in the fourth position as the sole positivelycharged residue was included in this study (Table 1); when that positive charge was converted to a neutral residue by acetylation the rate increased about 5-fold, and when it was converted to a negatively charged residue by succinylation, the rate increased nearly 7-fold. The negative effect of positive charges on the hydrolase is thus well documented, but the different rates observed with charged His, k g , and Lys suggest that the rate is affected by other features in addition to the positive charge. To further explore the basis for the negative effect of Pro and Arg in the third position of peptide substrates, kinetic analysis was carried out under the standard assay conditions with two pairs of I4C-Ac-peptides differing

kc,,

Krn

Substrate

(nmolhnin)

(mM)

Ac-AAFG Ac-AARG Ac-AAAA Ac-AAPA

23.9 1 .o 22.1 1.1

2.0 1.7 2.1 6.73.6

kc,,/Krn

11.9 0.59 10.5 0.3

Ki (mM)

1.6

only in the third residue. The assays were carriedout over a range of substrate concentration from 0.02 to 25.6 mM, approximating initial rate determination by selecting hydrolysis times giving mostly 5-7%, and never exceeding 12% conversionof the substrate. The results are reported in Table 3 and show that the low rates observed with the Arg- and Pro-containing peptides are primarily derived from an effect on the k,,,. The K, does not appear to be affected significantly. This typeof effect appears to be quite general for poor substrates for proteases. When I4C-labeled Ac-AAFG and Ac-AAAA were assayed in the absence and presence of ’H-labeled Ac-AARG and Ac-AAPA, respectively, analysis of the 14C-Ac-A produced showed the expected competitive inhibition by the “poor” substrates with Kivalues within a factor of 2 of the K,. Table 2 shows the rate constants for anumber of fairly longpeptidesubstratescontainingmultiple positive charges as well as negative charges. A major point to be illustrated by the data in Table 2 is that the activity of the enzyme in general decreases drasticallyas the peptide gets longer, but that certain fairly long peptides that contain negatively charged residues remain good substrates. In addition, the data for thethree sets of peptides from myelin basic protein confirm the negative effect of positive charges and the general observation that succinylation of Lys gives a more favorable substrate than does acetylation. The modified insulin chains have been included primarily because of their relevance to the question about the use of the hydrolase to unblock acetylated large peptides, or ideally intact proteins, for sequencing. In our hands the enzyme has not been useful for this function, but the fact that hydrolysis is observed at all with the 28-residue peptide from InsB may suggestthat under appropriate conditions with the proper modification of the substrate, this function may eventually be established. Regarding the application of the hydrolase to protein sequencing, it has been demonstrated that theenzyme can be used to unblock the acetylated N-terminus after fragmentation of the protein, blocking the new N-terminals either as phenylhydantoins (Sakiyama, 1990) or as succinyl amides (Krishna et al., 1991), or removing them by coupling to isothiocyanate-modified glass beads (Farries et al., 1991).

R.G. Krishna and F. Wold Discussion

apparent lack of activity toward various earlyintermediates. Based on thein vitro properties of the enzyme, short The acylaminoacylpeptide hydrolases have been purified nascent chains should be viable substrates for the enfrom rat liver (Tsunasawa et al., 1975; Kobayashi & zyme, unless some special features of primary structure Smith, 1987), bovine liver (Gade & Brown, 1978), pig render them resistant. The specificity determinants establiver (Mitta et al., 1989), human erythrocytes (Schonlished in this work may thus be among the unique features that contribute to the stability of the acetylated berger & Tschesche, 1981; Jones & Manning, 1985), and sheep erythrocytes (Witheiler & Wilson, 1972), and a good N-terminus in these proteins in vivo. The wealth of data deal of information about the molecular and catalytic available for the N-terminal sequences of acetylated proproperties of these enzymes as well as of the present musteins makes it possible to assess the specific proposition cle enzyme (Radhakrishna & Wold, 1989, 1990) has been that the presence of Pro and/orpositive charges in the Nreported. Although there aremany conflicts in detail, the terminal sequence of acetylated proteins contribute to specificities of the different hydrolase preparations have their stability. Persson et al. (1985) examined the initial 10 residues in a large number of acetylated proteinsand certain common features. Thus, therequirement for Thr, found that Lys, for example, is statistically overrepreAla, Met, Ser, andGly as theacetylated N-terminus apsented in these sequences. When the sameset of sequences pears to be general, and similarly the decreasing activity was reexamined to assess the presence of all of the proas the substratepeptides get longer also appearsto apply posed negative effectors, it was found that of a total of across the board. On the other hand, the negative effect of Arg reported for the muscle (Radhakrishna & Wold, 107 eukaryotic acetylated proteins, only 6 were without these residues. Positive charges were present among the 1990) and human erythrocyte(Jones et al., 1991) enfirst 10 residues in 84, and Pro in 26, and of these, 56 had zymes, appears to be in conflict with the reported unthe positive charges and 19 the Pro in the first 5 residues. blocking of the sequenceAc-Asp-Arg---- by the rat liver Although the score is not perfect, it certainly suggests that enzyme (Kobayashi & Smith, 1987), and there does not a relationship exists between stability and primary seappear to be any agreement on the possible stimulation quence and that Pro and positive charges are significant by negative charges observed in our studies. The possibilcontributors, probably along with other sequence feaity that the negatively charged C-terminus may have a sigtures, to the resistance to the hydrolase. Based on our renificantstimulatoryeffect on theenzymehas been sults, the best model for the active structure of a good considered and specifically evaluated as an explanation substrate for thehydrolase appears to be an N-terminal of the decreasing activity observed with increasing pepsequence in an extended chain conformation. Negative tide length. Data in theliterature suggest that the C-tercharges could favor this conformation in the active site minal charge is not an important factor, however. Thus, environment, whereaspositive charges, also favoring this in the case of Ac-AAA and Ac-AAAA the activity was conformation, could be incompatible with the active site reduced only 50% and 85%, respectively, when the C-tercharges. The effect of Pro can then be explained in terms minal carboxylate was converted to the methyl ester of deviation from the extended chain structure, causing (Gade & Brown, 1978). The recent cloning of the rat interference from the subsequent sequence extension. It (Kobayashi et al., 1989) and pig (Mitta et al.,1989) liver is tempting to propose an active site composed of multienzymes has demonstrated that the sequences of these ple subsites determining the role of the acylamino acid enzymes are very similar, and because our direct sequencand atleast the next two residues in forming the catalyting dataforthe muscleenzyme N-terminus (Acically active enzyme-substrate complexes. Such a model MERQVL-) (Krishna et al., 1991) matches that of the would be quite speculative at the present time and would liver enzymes, it is probably appropriateto consider the still leave many important features unresolved, notably possibility that all of these hydrolases represent a single the effect of Arg in positions 4-6 and theapparently difenzyme even if the data on themolecular make-up and ferent magnitude of the effect of His, Arg, and Lys at substrate specificity for the different hydrolases at this different points in the sequence. Establishing such a stage may suggest that a family of different enzymes model is clearly the ultimate goal of the present studies. exists. Other groups have studied the specificity of the hydroConsidering the hydrolase with the view that it coexlases, as well as of other enzymes involved in the N-terists with a number of N-acetylated proteins in the cytominal processing of proteins in general. The relationship sol of many different cells, it is clear that the acetylated between the N-terminal structure and protein turnover proteins in those cells must be resistant to the action of (Bachmair et al., 1986; Dice, 1987; Mayer et al., 1989) is the hydrolase. Because none of the hydrolase preparaa fascinating new development with great relevance to the tions to date have been found to have any activity against naturally acetylated proteins, thisview does not conflict whole area of how protein sequences are “read”in vivo, and how the messages encoded in the N-terminal sewith observed facts. However, if the enzyme indeed is quences are being translated into different types of biopresent throughout the biosyntheticlife of the different logical actions (Arfin & Bradshaw, 1988; Bradshaw, cytosolic proteins, there is no obvious explanation of its

587

Specificity of acylaminoacyl-peptide hydrolase

1989). At this stage it appears that the key enzymes in the cotranslational N-terminal processing of proteins are the Met aminopeptidase (Huang et al., 1987) and the acetyl transferases, of which there are two in yeast, one specific for Met (Lee et al., 1990) and another specific for the other amino acids (Lee et al., 1988). In acknowledging the exciting correlations between the nature of the N-terminal processing and protein turnover and the importance of the first and second residues in directing the processing steps, it must also be accepted that the total sequence information that goes into the regulation of these processes must be much more extensive. Recent studies on the rat liver polysome N-acetyl transferase illustrate this point well (Yamada & Bradshaw, 1991a,b). Not only do these studies demonstrate a direct effect of the third residue in the sequence, but they also clearly show that the effect of the second and third residues are different for different N-terminal amino acids and that the total sequence in some of the longer peptides influences the substrate quality in some yet undetermined manner. These latter points define the key challenges for future work; both the explanation of the apparent discrepancies in specificity reported from different laboratories and the rational description of the specificity determinants will require a far better understanding of all the information encoded in the N-terminal sequences. At this stage it seems reasonable to apply the concept of “context” when amino acid sequences are being considered as the basis for enzyme specificity; given sequences and the individual members of those sequences are obviously affected by the total environment in which they reside, and identity of chemical properties of two identical sequences can only be expected whenever their environments are identical. Just like a given combination of letters in a word has a specific meaning in a given sentence, but may take on a very different meaning aspart of a different word in a different sentence, a given amino acid sequence may well express its unique signal only in the context of one unique environment. This concept of context has become a very significant component in protein structure predictions inwhich statistical data from known three-dimensional structures are applied to predict the folded structures of known linear sequences (see Bowie et al. [1991] for a recent discussion), and it seems clear that it must becomean important consideration in exploring the unique reactivity of individual amino acid side chains and peptide bonds responsible for the exquisite specificity observed in posttranslational modification reactions. Materials and methods

Materials and general methods

Peptides were synthesized with the Applied Biosystems 430A Peptide Synthesizer or obtained from Research Plus

Inc., Bachem Bioscience Inc., and Peninsula Laboratories. 14C- and 3H-labeled acetic anhydride were purchased from NEN and ICN, respectively. All reagents were of reagent grade. Oxidized insulinchain A, chain B, myelin basic protein, carboxypeptidase A, carboxypeptidase B, trypsin, and chymotrypsin were either from Sigma or from Worthington Biochemical Corporation. Amino acid analyses were performed with the LKB Alpha Plus Amino Acid Analyzer, peptide sequencing with the Applied Biosystems 477AProtein Sequencer, and fast atom bombardment (FAB) massspectra (MS) weretaken on a Kratos MSSORF high resolution mass spectrophotometer. NO-Acylaminoacyl-peptide hydrolase, a cytosolic enzyme with native M, of 245,000 was purified 7,500-fold from young rabbit muscle (Pel-Freez) according to our earlier reported procedures (Radhakrishna & Wold, 1989). The enzyme is quite stable, showing an activity loss of about 5% over a span of 2 months when stored at -20 “C. The activity of the enzyme was monitored regularly, and all the assays reported were carried out with enzymepreparations having at least 95% of native specific activity. Protein assays were carried out according to the protein-dye method (Bradford, 1976) and the enzyme purity was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970). Evaluation of substrate quality

The enzyme assays were carried out according to published procedures (Radhakrishna & Wold, 1989). Routinely, 0.5pg of enzyme wasincubated at 37 “C with 1 mM substrate in 200 pL of assay buffer (50 mM [Na] phosphate buffer, pH 6.9, containing 1 mM EDTA and 2 mM MgC12). For the best substrates these conditions would result in hydrolysis of 5-25Vo of the substrate in 30 min, and the standard assay was incubated for 30 min before the reaction was stopped by heating at 100 “C for 5 min. For poorer substrates, the amount of enzyme was increased up to 10-fold and/or the incubation time was lengthened up to 4 h. The activity assays were always based on the determination of product produced and substrate remaining after separation of the two on high performance liquid chromatography (HPLC); for the I4C-labeledsubstrates, this was readily done by direct assay of the radioisotope. The identification of the radioactive product was always confirmed by coelution with the appropriate 3H-acetylamino acid derivative, added to the reaction mixture prior to HPLC. For the comparison of the different substrates the rate constants have been normalized to reflect the rate with 1 pg of enzyme. For a few nonradioactive substrates, isolated as acetylated N-terminal peptides from naturally acetylated proteins, the substrates and products could be detectedby their 230-nm absorbance, and quantification of the appropriate fractions from HPLC (Fig. 2) was accomplished by acid hydrolysis and amino acid analysis, using norleucine as internal standard.

R.G. Krishna and F. Wold

81

53-

n PI

n

nn p:

3 P4

..'

ln

1 Retention Time (min) Fig. 2. The detection of substrates and products of the hydrolase reaction in the absence of radioisotopes. A series of peptides were prepared from the acetylated N-terminus of myelin basic protein, and the Lys residues were either left intact or modified byacetylation (indicated by J) or succinylated (indicated by U). The hydrolase digests of these substrates were fractionated on a C-18 (100-A) column with a 5-min isocratic elution of 0.1'70 TFA followed by a 35-min linear 0-40'70 gradient of CH&N containing 0.1'70 TFA. The elution positions of the different substrates and products are indicated asfollows: S1, AcAAQK, Ac-AAQJ, Ac-AAQU; S2, Ac-AAQKRPS, Ac-AAQJRPS, Ac-AAQURPS;S3,Ac-AAQKRPSQRSKY,Ac-AAQJRPSQRSJY, Ac-AAQURPSQRSUY;S4,Ac-AAQKRPSQRSKYLASASTX,ACAAQJRPSQRSJYLASASTX, Ac-AAQURPSQRSUYLASASTX (X denotes the mixture of homoserine and homoserine lactone). The product peaks P1, P2, P3, and P4 indicate the corresponding peptides after removal of Ac-Ala.

monitoring the absorption at 230 nm. The 95% pure N-terminal peptide thus obtained was further purified by HPLC ona C-18 (300-A)reverse-phase column with a 25-min, 10-35% gradient of CH,CN containing 0.1% trifluoroacetic acid (TFA),into 0.1% aqueous TFA, yielding two peaksthat were characterized by amino acid analysis and FAB-MS as the free acid and lactone forms of theN-terminalfragment, Ac-AAQKRPSQRSKYLA SASTX (X = homoserine or homoserinelactone)with calculated/observed (M + H+) of 2,094.2/2,094.4 and 2,076.4/2,076.4, respectively.An aliquot of 5 nmol of the mixture of the two peaks gave zero background on sequencing, confirming the absence of a free N-terminus. In a separate preparation, 75 mg (4 pmol)of starting material was used to prepare the CNBr peptide as precursor for three additional N-terminally blocked peptides, AcAAQKRPSQRSKY, Ac-AAQKRPS, and Ac-AAQK. These were prepared from the parent peptide by digestion with different proteases and isolation of the products on either 100-A or 300-A size C-18 columns by reverse-phase HPLC with the aqueous TFA-CH3CN gradient. All the major peaks observed on HPLC from each protease digest were characterized by amino acid analysis and FAB-MS. First the CNBr peptide was digested with chymotrypsin in 0.08 M Tris-HC1 buffer, pH 7.8, containing 0.1 M CaClz with an enzyme substrate ratio of 1:50 for 2 h.An aliquot of the reaction mixture was acidified with TFA and subjected to purification by HPLC (C-18 [300-A] column with a 2O-min, 5-30% gradient) to yield Ac-AAQKRPSQRSKY (calc. [M H+], 1,462.6; found, 1,461.8). The remainder of the chymotryptic digest was next heated (5 min, 100 "C) and treated with carboxypeptidase A and B at enzyme substrate ratios of 1:20 and 150, respectively, for 2 h. The digest was acidified and purified on a C-18 (100-A) column witha 25-min, 5-30% gradient to yield Ac-AAQKRPS (calc. [M H+], 799.9; found, 799.4). Ac-AAQK (calc. [M H+], 459.5; found, 459.3) was prepared by digesting Ac-AAQKRPS QRSKYLASASTX with trypsin in 0.04 M Tris-HC1 buffer, pH 8.1, containing 0.05 M CaC12 for 2 h with an enzyme/peptide ratio of 1:20. The digest was acidified and eluted on a C-18 (100-A) column with a 25-min, 530% gradient. About one-third (0.4 pmol) of each of the four products was kept to be used as substrate for hydrolase, and the remaining material was subjected to chemical modification to alter the charges on the peptides. The €-amino function of the Lys residues in the four peptides was either neutralized by acetylation or converted to a negative charge by succinylation. The acetylation with acetic anhydride was carried out as previouslydescribed; the succinylation was carried out atpH 8.0 in 0.4 mL of 1 M carbonate buffer by treatment with a 100-fold excess of succinic anhydride in dioxane; the reagent was added in multiplealiquots with stirring at room temperature and the pH was maintained at 8.5 with the addition of 0.1 N NaOH. The lyophilized products were desalted by HPLC

+

+

Preparation of substrates The common method for the preparation of acetylated peptides was either through the direct acetylation of peptides with I4C-acetic anhydride or through the reaction with the N-hydroxysuccinimide esters of 14C-acetylamino acids or I4C-acetyldi-and tripeptides. The details have been described elsewhere (Radhakrishna & Wold, 1990). All the products were characterized by FAB-MS and HPLC. To prepare a family of fairly complex peptides containing positive chargesand Pro, the N-terminal cyanogen bromide (CNBr) peptide of myelin basic protein was isolated and further modified by specific proteolysis as follows: bovine myelin basic protein (25 mg, 1.35 pmol) was treated with CNBr according to the procedures described in the literature (Gross, 1967). The reaction products were lyophilized, redissolved in 0.5 mL of 0.5% acetic acid, and fractionatedon a 160 x 1.2-cm Sephadex G-50 column with a fraction size of 2.8 mL,

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589

Specifcity of acylaminoacyl-peptide hydrolase essentially under the same conditions that were used for the purification of the unmodified peptides. The calculated and the observed (M H+) of NE-acetyl- (indicated by J) and Ne-succinyl- (indicated by U) derivatives are given in parentheses; Ac-AAQJRPSQRSJY (1,546.6/ 1,546.8), Ac-AAQURPSQRSUY (1,662.6/1,662.0), ACAAQJRPS (841.8/841.4), Ac-AAQURPS (899.8/899.4), Ac-AAQJ (501.5/501.4), and Ac-AAQU (559.5/559.3). Another set of large peptide substrates was prepared by reacting oxidized insulin chain A (InsA) and des-Lys, Ala-insulin chain B (desKA-InsB). Prior to use in these reactions, the oxidized InsB wastreated with carboxypeptidases A and Bto remove the penultimate Lys residueas an additional acylation site. An aliquot of each peptide was acetylated with acetic anhydride and other aliquots were acylated with the hydroxysuccinimide estersof 14Clabeled Ac-Ala, Ac-Met, and Ac-Gly, respectively (Radhakrishna & Wold, 1990). FAB-MS showed that all the products had been overacylated, as should be expected because both chains contain two Tyr residues, and the products were consequently treated with 0.5 M alkaline hydroxylamine (pH 10) for 28 h at 30 "C to eliminate the putative esters. Milder conditions in terms of hydroxylamine concentration, pH, or reaction temperature and time, which have been used for cleaving ester linkages in proteins in the past (Olson et al., 1985), gave onlypartial removal of the excess acylaminoacyl groups; under the conditions described, the reaction went essentially to completion. The pooled product peaks from HPLC were lyophilized and characterized by FAB-MS, givingthe following calculated/experimental (M H+) ratios: oxidized 1nsA:Ac-, 2,574.0/2,574.6; Ac-A-, 2,644.1/2,643.0; Ac-M-, 2,705.2/2,706.0; Ac-G-, 2,631.1/2,632.8; oxidized desKA-1nsB:Ac-, 3,338.7/3,339.4; Ac-A-, 3,409.U 3,411.4; Ac-M-, 3,469.9/3,471.7; Ac-G-, 3,395.8/3,395.2.

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Acknowledgments This work was supported in part by grants from theRobert A. Welch Foundation, AU-916 and AU-0009, and from the United States Public Health Service, GM31305.

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