Proc. Nati. Acad. Sci. USA Vol. 88, pp. 11510-11514, December 1991 Biochemistry

A rapid method for determination of endoproteinase substrate specificity: Specificity of the 3C proteinase from hepatitis A virus (proteinase/Edman degradation/peptide mixures)

JOANNE R. PETITHORY*, FRANK R. AND BRUCE A. MALCOLM II

MASIARZt#, JACK F. KIRSCH*, DANIEL V. SANTI§,

*Department of Molecular and Cell Biology, University of California, and Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720; tChiron Corp., Emeryville, CA 94608; *Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143; §Departments of Biochemistry and Biophysics, and of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143; and lProtos Corp., Emeryville, CA 94608

Communicated by Thomas C. Bruice, September 9, 1991

The preferred amino acid residues at the P; ABSTRACT and P2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P' or P2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termin, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative k../Kt. values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P; mixture were glycine, alanine, and serine. The enzyme has little specificity at P2; only argnine and prolin peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.

Many proteinases have specificities that extend several residues on either 'side of the scissile bond (1). The general problem of elucidation of the preferred substrate sequences therefore requires the synthesis and kinetic analysis of numerous peptides of defined sequence-a time-consuming and expensive process. We report here a rapid method for the quantitative determination of endoproteinase substrate specificity for subsites on the C-terminal (P')** side of the scissile bond of a peptide substrate that largely circumvents these limitations. This method'uses the proteinase-catalyzed competitive hydrolysis of defined peptide mixtures to map the subsite preferences of the proteinase. The' substrate is a mixture of N-acetylated peptides, identical in sequence except for a single position. The strategy is outlined in the example shown in Fig. 1, for a mixture containing three different amino acids in" the degenerate position. At a given time during the reaction, peptides may be unhydrolyzed, partially hydrolyzed, or completely hydrolyzed depending on their sequence. The specificity is determined by product analysis without further purification. The 3C proteinase from hepatitis A virus (HAV-3C) is a cysteine ertdoproteinase that cleaves the viral polyprotein to mature viral proteins (reviewed in refs. 3-5). The 3C proteinases from various picornaviruses, such as poliovirus, human rhinovirus, encephalomyocarditis virus, as 'well as HAV, have been shown to cleave peptides that correspond in

sequence to known or predicted polyprotein processing sites (refs. 6-9; B.A.M., S. M. Chin, J. R. Stratton-Thomas, K. B. Thudium, R. Ralston, S. Rosenberg, and D. A. Jewell, unpublished data). The exact determinants of 3C proteinase specificity are not well understood. In poliovirus, each of the eight 3C-specific polyprotein cleavage sites occurs between glutamine-glycine bonds; however, not all glutamine-glycine pairs in the polyprotein are cleaved, suggesting additional sequence or conformational determinants. In' other picornaviruses, the known or predicted polyprotein cleavage sites are not limited to glutamine-glycine pairs; the P' residues are replaced in some picornaviral polyproteins by serine, alanine, methionine, threonine, or valine (3, 4). Proteolysis of the polyprotein by 3C proteinase is essential for viral replication (10-14). It should be feasible to exploit the specificity of these proteinases to develop 3C proteinase inhibitors as antiviral agents (15, 16). An important step in the design of such highly specific 3C proteinase inhibitors is the delineation of the specificity of the proteinase. Since nature has provided only a small set of the amino acids present at various positions, we have undertaken a study using mixtures of synthetic peptides to explore a more complete set of substitutions. This method is generally applicable to the study of the specificity of endoproteinases and enzymes whose active sites have been engineered for use in specific sequence synthesis and degradation.

MATERIALS AND METHODS HAV-3C Proteinase. The HAV-3C proteinase was overexpressed in Escherichia coli in a soluble and active form. The protein was expressed intracellularly upon induction with isopropyl /3-D-thiogalactopyranoside, with a final yield after purification of 15 mg per liter of culture. A detailed description of the expression and purification of the HAV-3C proteinase will be presented elsewhere (B.A.M., S. M. Chin, J. R. Stratton-Thomas, K. B. Thudium, R. Ralston, S. Rosenberg, and D. A. Jewell, unpublished data). Peptide Synthesis. Defined sequence peptides. These substrates were synthesized by standard solid-phase 9-fluorenylmethyloxy carbonyl chemistry (17) and acetylated at the N termini with acetic anhydride. The peptides were cleaved from the Rink resin (18) supports, using trifluoroacetic acid (TFA) and appropriate scavengers, to generate C-terminal Abbreviations: HAV, hepatitis A virus; PTH, phenylthiohydantoin. "To whom reprint requests should be addressed at: Chiron Corp., 4560 Horton Street, Emeryville, CA 94608. **The nomenclature of Berger and Schechter (2) is used. Amino acids are numbered consecutively away from the scissile bond, P, and P denoting the N- and C-terminal sides, respectively. The corresponding enzyme subsites are denoted Sn and Sn.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

11510

Biochemistry: Petithory et al. x

I I

x4

I I

x

R

4

xi

I

I

x4

I

x

4

x

I I

I

I

I

FA

I

I

I

x4

x4

4

11511

Peptide kcat/Km

x4

x 4M x4

Proc. Natl. Acad. Sci. USA 88 (1991)

-I

E3

+

*

++

I

--I -

FIG. 1. Representation of the partial proteolysis of a peptide mixture containing three different amino acids in the degenerate Pi position. The site of proteolysis is indicated by the arrowhead. Peptides containing amino acid substitutions a, and * are poor, fair, and good substrates, respectively. At a given time during the reaction, peptides may be unhydrolyzed (c), partially hydrolyzed (a), or completely hydrolyzed (-) depending on their sequence. Quantitation of the extent of hydrolysis of each component in the mixture is accomplished by direct sequence analysis via N-terminal Edman degradation, without prior separation of reactants from products. Neither the uncleaved peptides nor the N-terminal halves of the cleaved peptides are quantitated because of the acetylated N termini (x-). r,

amides, purified by reversed-phase HPLC [C-18, 5 x 25 cm; Vydac (Hesperia, CA), 1%/min linear gradient; A, 0.1% TFA/water; B, 0.1% TFA/acetonitrile] and characterized by amino acid analysis. Peptide mixtures. Two peptide mixtures were prepared by a modification of the methods described (19) with the general sequence Ac-Nle-Glu-Leu-Arg-Thr-Gln-P;-P'-Ser-Asn-ArgNH2. Phenylalanine occupied the P' position in the P' mixture and serine occupied the Pi position in the P' mixture. The mixtures were synthesized by standard solid-phase tertbutyloxycarbonyl (tBoc) chemistry on methoxybenzhydrylamine resin (20). The standard protocols provided for the Applied Biosystems model 430A peptide synthesizer were followed for all couplings, except for the degenerate position, where a 10-fold molar excess over resin of an equimolar mixture of 17 tBoc amino acids was used. The coupling mixtures contained norleucine (for methionine) and all of the naturally occurring amino acids except glutamine (to avoid alternate cleavage sites), tryptophan, and cysteine. Arginine was placed at the C terminus to aid in disk retention during Edman degradation. The full-length mixtures were acetylated at the N termini using acetic anhydride. After coupling of glutamine to the P' mixture, an aliquot of the resin was removed and the peptide was cleaved from the resin, resulting in the unblocked P' control peptide mixture Gln-P;-Phe-Ser-Asn-Arg-NH2. After coupling of the second serine to the P' mixture, an aliquot of the resin was removed and the peptide was cleaved to give the unblocked P' control mixture Ser-P'-Ser-Asn-Arg-NH2. Both the full-length acetylated mixtures and the unblocked control mixtures were deprotected and cleaved from the resin with hydrogen fluoride (20). The mixtures were desalted on reversed-phase HPLC columns and characterized by amino acid analysis. The control mixtures were also analyzed by Edman degradation. Proteinase Assays. The extent of proteolysis of individual peptides was quantitated by fluorescamine labeling (21) ofthe N termini generated by peptide hydrolysis. Reaction mixtures contained 100 mM sodium phosphate (pH 7.6), 5 mM dithiothreitol, 2 mM EDTA, 10% (vol/vol) glycerol, 50-200 ,M peptide, and 24-36 gg of 3C proteinase per ml (1.0-1.5 AM) and were incubated at 370C. Aliquots (10-50 juI) were quenched at selected time points by dilution into 2.5 ml of 0.2 M sodium borate (pH 8.6), with vigorous mixing. Fluorescamine (0.25 ml of 0.3 mg/ml in acetonitrile) was added with vigorous mixing. Fluorescence emission was monitored at 475 nm with excitation at 390 nm. The fluorescence emission readings were converted to primary amine concentrations from a standard curve constructed from the peptide Ser-PheSer-NH2. Fluorescence yields were linear with concentration to at least 50 jiM. Initial velocities were linear to at least 20% hydrolysis.

Proteolysis of Peptide Mixtures. Reaction mixtures contained 100 mM sodium phosphate (pH 7.6), 5 mM dithiothreitol, 2 mM EDTA, 10% (vol/vol) glycerol, 450 jiM P1 mixture or 650 AiM P' mixture, 12.5 jig of 3C proteinase per ml (0.5 jiM) and were incubated at 370C. Aliquots were withdrawn 20 min, 1 hr, and 4 hr after enzyme addition, quenched with an equal vol of 50%o acetic acid, and stored at -200C for sequence analysis. The extent of proteolysis was monitored by fluorescamine assays as described above. Sequence Analysis of Peptide Mixtures. Automated Edman degradation was performed on Applied Biosystems model 470A or 473A protein sequencers equipped with model 120A on-line phenylthiohydantoin-derivatized (PTH)-amino acid analyzers using the programs and reagents supplied by the manufacturer. Quenched aliquots from the proteolysis mixtures, containing 4 nmol of liberated amine (by fluorescamine assay), were loaded onto Polybrene disks (equal amounts of product were analyzed to maximize sensitivity and reproducibility). Two 30-sec manual deliveries of ethyl acetate interspersed with 30-sec deliveries of argon were used to remove glycerol before initiation of the run. Two aliquots from each of the time points were sequenced in duplicate. The yield of each PTH-amino acid in the sequencing cycle corresponding to the degenerate position of the mixture (cycle 1 for the Pi mixture and cycle 2 for the P' mixture) was used to quantitate peptide product. Later cycles were used to confirm the specificity of the cleavage site. Quantitation of PTH-Amino Acids. The integration software was calibrated with a PTH-alanine standard. The 6269 = 16,000 M-1lcm-1 of PTH-alanine (22) was used in conjunction with the extinction coefficients of the other PTH-amino acids (22) to calculate the yields of the Edman degradation products. This method precludes the need to inject individual standards for each PTH-amino acid. Several assumptions were made to simplify the quantitation approach. The 1040% acetonitrile gradient was assumed not to affect the extinction coefficients of the PTH-amino acids; the extinction coefficients of the PTH-lysine and diphenylthiourea were assumed to be equal, as well as those for PTH-serine and the adduct of its dehydration product with dithiothreitol (PTH-serine). Manual injection of 50 pmol of the standard determined detector response and peak area in terms of AiV-sec. A response factor (RF) for PTH-alanine was established as jiV-sec/pmol. The response factors for the other PTH-amino acids were derived from the equation RFxaa

=

RFAia

-

e269Ala

A comparison of the yields of each amino acid made accessible by proteolysis to the total available in the control peptide mixture, which was not blocked by acetylation, made

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it possible to estimate the percentage proteolysis by Edman degradation and to avoid the major pitfalls in the quantitative analysis of amino acid sequence data (i.e., differences in initial and repetitive yields, differential extraction, conversion and destruction of amino acid derivatives, and variable recovery of PTH-amino acids during reversed-phase chromatography). Determination of Relative kwtlK Values. The yield of each amino acid from sequence analysis was used to calculate the total mol of each product peptide formed in the reaction. This together with the calculated mol of each substrate peptide based on the Edman degradation of the control mixtures allowed the calculation of the molar percent of each component proteolyzed at a given time. This was plotted versus time. The slopes and standard errors were determined by linear regression. These slopes gave the relative velocity (v) for each peptide in the mixture. The kcat/Km value for each peptide was then determined, relative to that of the mixture component bearing the sequence Ser-Phe in the Pj-P2 positions, using Eq. 1, VA VB

kcat/Km[A] kcat/Km[B]

(23) where A corresponds to a given mixture component and B corresponds to the Ser-Phe peptide. The molar percentages of the mixture components (Table 1) were used for the concentration terms.

RESULTS Preparation of Peptide Mixtures. Peptide mixtures were synthesized by coupling an equimolar mixture of amino acids at either the P' or P' position of the substrate peptide. Aliquots were removed one cycle after coupling the amino acid mixture to obtain control mixtures amenable to sequence analysis. The relative amounts of each component in the Pi and P2 control mixtures (i.e., with free N termini) as determined by Edman degradation, are given in Table 1. Amino acid analysis was performed on each mixture and values obtained for the unique amino acids were in good agreement with those found by sequence analysis (data not shown). The concentrations of the individual peptides in the mixtures varied over a 10-fold range. Arginine and histidine in the Pi mixture, and histidine in the P2 mixture, were not present in sufficient amounts for reliable quantitation and these mixture components were omitted from further analysis. As both the control mixtures and the experimental mixtures were derived from the same synthesis, it was assumed that the relative amounts of each component in both mixtures were the same. Proteolysis of Peptide Mixtures. Table 1 shows the sequences of the peptide mixtures used in this study. The parent sequence (Ac-Nle-Glu-Leu-Arg-Thr-Gln-Ser-PheSer-Asn-Arg-NH2) is based on that of the putative 2B/2C

Proc. Natl. Acad Sci. USA 88 (1991) 1 00

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0)a: 0)

0L

2 4 3 Time (hours)

6

FIG. 2. Proteolysis of PI and P2 mixtures catalyzed by the HAV-3C proteinase. Reaction conditions were 100 mM sodium phosphate, 5 mM dithiothreitol, 2 mM EDTA, 10%o glycerol, 0.45 mM P1 mixture (*) or0.65 mM P2 mixture (-), and 12.5 jig ofHAV-3C proteinase per ml (pH 7.6) at 37TC. Reaction progress was monitored with fluorescamine.

cleavage site of the HAV polyprotein (24). Initial tests of HAV polyprotein cleavage site peptides suggested that the 2B/2C sequence was rapidly proteolyzed by the 3C proteinase (W. Swietnicki and B. M. Dunn, personal communication). The time courses for the proteolysis reactions of the P' and P2 N-acetylated peptide mixtures are shown in Fig. 2. The reactions were essentially complete after 4 hr. The extent of total product formation was ;35% for the P' mixture and =75% for the P2 mixture. Samples were removed from the reaction mixtures at various times, quenched in acetic acid, and sequenced to ascertain the relative amounts of cleavage of each component in the mixture. A graphic display of the results for 8 of the 15 component peptides of the P1 mixture is shown in Fig. 3. Relative km/Km Values. The relative kcatlKm values were calculated from Eq. 1, which applies to a reaction in which 2 or more substrates compete for the enzyme, and are shown

0.10

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Q s°w

0

0.06

P-

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Table 1. Composition of mixtures with the general sequence

Ac-Nle-Glu-Leu-Arg-Thr-Gln-Pj-P2-Ser-Asn-Arg-NH2

Pi mixture P2 mixture Asp (11.4) Pro (6.6) Asp (4.4) Arg (8.6) Asn (3.7) Pro (8.4) Asn (6.0) Val (3.1) Ser (7.1) Phe (10.4) Ser (2.4) Val (4.6) Thr (1.2) Ile (2.0) Thr (1.7) Phe (9.4) Gly (8.9) Lys (4.0) Gly (9.3) Ile (3.2) Glu (6.8) Lys (3.0) Glu (9.7) Leu (7.1) Ala (11.8) Nle (3.6) Ala (13.8) Leu (8.9) Tyr (6.7) Tyr (7.0) Nle (4.8) Molar percent of each component is shown in parentheses. Quantitation is by sequence analyses of unblocked control mixtures. Pi, phenylalanine; Pf, serine.

0.02

0.00

Time (Hours) FIG. 3. Initial rates of proteolysis of the individual component peptides of the Pi mixture after reaction with HAV-3C proteinase. Data for 8 of the 15 individual component peptides of the PI mixture are shown. Most ofthe data points for the alanine, serine, and glycine components are not visible because of the expanded scale.

Biochemistry: Petithory et al. 1.2

Proc. Natl. Acad. Sci. USA 88 (1991)

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Amino Acid at Position P1'

FIG. 4. Relative k'(at/Km values of the PI mixture after reaction with HAV-3C proteinase. Velocities were obtained from slopes ± SE of the plots of the amount proteolyzed versus time and corrected for concentration. Corrected velocities were normalized to that of the peptide containing serine in the PI position to give the relative kcat/Km for each mixture component. Reaction conditions were the same as those described in Fig. 2.

in Fig. 4 for the P' mixture. The peptides containing the small, uncharged unbranched side chains of alanine, serine, and glycine in the Pj position are clearly the preferred HAV-3C proteinase substrates. Other allowed, but significantly poorer, substrates have tyrosine, the methionine analog norleucine, phenylalanine, asparagine, and valine in the Pi position. The peptides containing other amino acids in this position were cleaved at much slower rates. The relative kcat/Km values of the 16 substituent peptides in the P2 mixture are shown in Fig. 5. Contrasting with the strong Pi preference is the lack of proteinase selectivity for the P' position except for the clear exclusion of arginine and proline. Confirmation ofRelative ke,/K. Values. The validity of the method to determine relative kcat/Km values was verified by independent measurement of kinetic parameters of proteolysis of individually synthesized peptides. The peptide sequences were selected to cover a wide range of k'cat/Km values based on the results shown in Figs. 4 and 5. For example, the peptides bearing alanine in the Pi position or aspartic acid in the P2 position should have kcat/Km values similar to that of the parent peptide containing the "native" 1.6 E

1.2

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0)

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)

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Amino Acid at Position P2'

FIG. 5. Relative k'(/Km values of the P2 mixture after reaction with HAV-3C proteinase. Relative k'cat/Km values are normalized to that of the peptide containing phenylalanine in the P2 position, as described in the legend to Fig. 4 and in Materials and Methods.

11513

Table 2. Relative kcat/Km values predicted from competitive proteolysis experiments and from direct measurement Relative kcat/Km Mixture* Sequence* Individualt Ac-LnELRTQSFSNR-NH2 1.0 1.0 QAF 1.0 0.8 Pi, QYF 0.2 0.1 pi 0.1 QVF 0.1 Pi QSD 1.3 0.7 P2' QSP '0.01 0.004 P2' Amino acids are identified by the single letter code; Ln, norleucine. Pj to P2 positions are indicated in boldface. The uppermost sequence (where Pj to P2 is QSF) corresponds to the putative 2B/2C HAV polyprotein cleavage site (2). Peptides are identical in sequence except as indicated. *From Figs. 3 and 4 (i.e., mixture proteolysis). tMeasured by fluorescamine assay (i.e., individual peptide proteolysis), normalized to the 2B/2C peptide (PI to P2 = QSF). Assay conditions were the same as in Fig. 2.

2B/2C sequence. Substituting tyrosine in the P' position, however, should yield a sequence of lower reactivity, while a peptide with proline in the P2 position should be resistant to proteolysis. Table 2 shows the sequences of the individual peptides synthesized and the relative kca/Km values obtained from the proteinase assays described above. These values are in good agreement with the relative kcat/Km values derived from the competitive proteolysis experiments. These results confirm the utility of competitive proteolysis to estimate rapidly the relative k'cat/Km values for the individual components of peptide mixtures.

DISCUSSION A procedure is described for rapid determination of the preferred amino acid residues in defined positions of endoproteinase substrates and has been applied to the study of the substrate specificity of the HAV-3C proteinase. The preferred substrates were selectively hydrolyzed by the enzyme from a mixture of peptides competing for the active site. The use of peptides acetylated at the N terminus permitted direct sequence determination of the preferentially cleaved peptides without prior separation from uncleaved substrates and the rapid estimation of kcat/Km for each peptide component in the mixture. Subsequent synthesis and characterization of individual peptides confirmed these initial estimates. The putative processing sites in the HAV polyprotein precursor cleaved by the 3C proteinase have been inferred by Wimmer and colleagues (24) from homologies to the known cleavage sites on the closely related poliovirus polyprotein. The P' positions of these putative in vivo HAV polyprotein cleavage sites include glycine, serine, methionine, and valine. The reaction of the HAV-3C proteinase with a peptide mixture containing amino acid substitutions in the Pi position (Fig. 4) resulted in the hydrolysis of peptides containing these same P' residues (with norleucine substituted for methionine), with a range of relative kcat/Km values of 0.1-1.0. In addition, the results indicate that alanine is a highly favored substitution in the P' position, which was not apparent from the polyprotein cleavage sites. It appears that small, uncharged, unbranched side chains are preferred in the PI position of HAV-3C substrates, but large residues such as tyrosine and phenylalanine can also be tolerated. The predicted P' positions of the HAV polyprotein cleavage sites are valine, threonine, phenylalanine, methionine, glycine, isoleucine, and glutamine according to Krausslich and Wimmer (3). The reaction of the HAV-3C proteinase

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Biochemistry: Petithory et al.

with a peptide mixture containing substitutions in the P2 position (Fig. 5) resulted in identification of all of the predicted amino acids (except for glutamine, which was not included in the mixture). Seven additional amino acids not suggested by the polyprotein cleavage sites-alanine, serine, tyrosine, leucine, aspartic acid, asparagine, and lysine-were also discovered to be good replacements at the P2 position, while inclusion of arginine or proline at this position yielded peptides resistant to proteolysis. Why the proteinase should discriminate against these two particular amino acids is unclear. It is worth noting, however, that, although proline is not an allowed substitution in the P' position of a HAV substrate peptide, it is commonly found in the P2 position of poliovirus and rhinovirus polyprotein cleavage sites; when this substitution is incorporated into peptide substrates for these enzymes, enhanced cleavage rates have been noted (3, 15, 16). There are 91 glutamine residues in the HAV polyprotein. Examination of these positions in light of the preferences deduced from the study described above would suggest at least 18 potential cleavage sites in addition to the 4 that were identified by homology with the poliovirus polyprotein (24) that also meet these criteria. The fact that these potential sites do not appear to undergo cleavage in vivo may be the consequence of (i) unidentified primary sequence requirements on the P side of the scissile bond, (ii) inaccessibility of the scissile bond to the proteinase, or (iii) absence or distortion of an essential tertiary conformation. The results described here from short peptide substrates confirm and expand on the P' and P2 specificity predictions for the HAV-3C proteinase (2). However, they may not directly reflect the sites of in vivo processing of the polyprotein mediated by the 3C proteinase, since the tertiary structure assumed by the polyprotein may contribute to the specificity in vivo (25-27). Both in vitro translation and in vivo replication methods have been used to probe the specificity requirements of 3C proteinases with regard to their polyproteins (3). The results of such studies in the poliovirus system, when compared with relevant peptide experiments (28, 29), suggest that peptide studies are in general, however, reasonable predictors of 3C proteinase behavior in vivo (3, 25, 30, 31). It should be noted that even with peptide substrates, the amino acid preference at any particular position may also be highly dependent on the nature of the side chains one, two, or even several positions away on the peptide (32). Only the effects of single-site substitutions within a peptide backbone were examined in this work. However, through a judicious choice of amino acid mixtures at several positions, this method could be used to evaluate the combinatorial effects of multiple substitutions in the P' positions. This would allow the scanning of extremely large sets of peptides that heretofore had been impossible by individual syntheses. By the incorporation of other analytical techniques, such as mass spectrometry, proteolysis of mixtures can be applied to the analysis of substrate specificity on the P side of the scissile bond as well. Such experiments are necessary to further refine methodologies and elucidate the substrate specificity of the HAV-3C proteinase. Note. After this manuscript was submitted for review, a similar strategy was published by A. J. Birkett et al. (33).

We thank Simon Ng for assistance in peptide preparation and Janice Kerr, Drs. Reyna Simon, and Steven Rosenberg for valuable discussions. This work was supported by Protos Corp. and by the

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A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.

The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been de...
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