Rapid optimization of enzyme substrates using defined substrate ...

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267 No. 3 Issue of January 25 pp. 1434-1437 1992 0 1992 by The American Sociky for Biochemistry and Mblecular Biolo&, Inc. Printed in U.S. A.

Rapid Optimization of Enzyme Substrates Using Defined Substrate Mixtures* (Received for publication, August 7, 1991)

Judd Berman$,Michael Green$, ElizabethSugg$, Rob AndereggS, David S. Millingtonp, Daniel L. Norwood$, J e r r y McGeehanS, and Jeffrey Wiseman$ From SGlaxo Research Laboratories, Research Triangle Park, North Carolina 27709 and the §Division of Pediatric Genetics and Metabolism, Duke University Medical Center, Durham, North Carolina 27710

of substrate specificity. This method relies on the ability to synthesize defined mixtures of amino acids at each subsite of peptide substrates,readily assignstructure toeach component in the mixture, and subsequently carry out enzymatic screening of the mixtures. Structural assignment is greatly facilitated by the use of combined high performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry (HPLC/CF-FAB-MS) (2-4). As an example of the utility of the method, we describe optimization of the Pi and PI positions of the collagenase substrate mimic Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH* (5-7). This substrate is cleaved specifically at the Gly-Leu linkage to yield Dnp-Pro-Leu-Gly-OH and H-Leu-Trp-AlaD-Arg-NH,. Usingthistemplate,threedifferentsubstrate mixtures were generated at both the Pi and P; positions, utilizing naturally occurring L-amino acids,D-amino acids, or miscellaneous unnatural amino acids, representing the synthesis of 128 different synthetic substrates. Results of the analyses of each of these subsites are reportedbelow.

A strategy is described for the rapid optimization of kCat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enEXPERIMENTALPROCEDURES hanced kCat/K,.High performance liquid chromatogPeptide Synthesis-The protected peptides with mixtures at the raphy/continuous flow fast atom bombardment mass Pi and P; positions were prepared starting with 0.5 mmol of p spectrometry is used to assign structure to each peak methylbenzhydrylamine resin (1.1 mmol/g) (Peptides International, in the chromatogram. As an example of the utility and Louisville, KY). The residues with fixed assignments were double efficiency of "substrate mapping"we describe optimi- coupled (preformed hydroxybenzotriazole esters in N-methylpyrrolization of the collagenase substrate Dnp-Pro-Leu-Gly- done) followed by capping with acetic anhydride. For coupling the Leu-Trp-Ala-D-Arg-NH,(where Dnp is dinitrophenyl) mixed residues, an equimolar amountof each of the aminoacids (0.25 at the P: and Pi positions. Six different mixtures were mmol total) wasconverted to the hydroxybenzotriazole ester and prepared for evaluation, representing the synthesis of coupled using an extendedcycle (1h) followed by exhaustive capping acetic anhydride. Trifunctional amino acids were protected as 128 different synthetic substrates. "Substrate map- with follows: N"-Boc-Cys(MeBz1)-OH, Ne-Boc-Asp(Chx)-OH, N"-Bocping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-DGlu(Chx)-OH, N"-Boc-His(Dnp)-OH, N"-Boc-Lys(2-Cl-Z)-OH, N"Arg-NHz, a substrate that possesses a 10-fold better Boc-Arg(Tos)-OH, Ne-Boc-Ser(Bz1)-OH, N"-Boc-Thr(Bz1)-OH,N"kCat/Kmthan Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-Boc-Tyr(2-Br-Z)-OH, N"-Boc-Orn(Cbz)-OH, N"-Boc-(p-aminoNHZ. Cbz)Phe-OH. Ne-Boc-Asnwas used in the mixture as its preactivated

p-nitrophenyl ester derivative. After the final coupling and N"-Boc deprotection, the resins were neutralized and treated with 2,4-dinitrofluorobenzene (0.55 mmol) anddiisopropylethylamine (0.55 mmol) in N-methylpyrrolidone (10 ml) for 3 h. The resins were then treated Substrateoptimization is a criticalstepin providinga with thiophenol (His(Dnp) removal), and the resulting resins were reliable and convenient enzyme assay and canprovide infor- cleaved using anisole (2 ml) in anhydrous H F (10 ml) a t -10 "C for mation for the optimization of inhibitor structure. The opti- 40 min. T h e H Fwas removed in vacuo, and theresidues were diluted mization problem is pronounced when searching for appro- with trifluoroacetic acid (50 ml) andfiltered. The residues were then priate substrates for proteolytic enzymes. Model substrates additionally rinsed with trifluoroacetic acid (2 X 50 ml). The trifluoare usually shortenedpeptidesegments derivedfrom the roaceticacid extracts were concentratedin vacuo, diluted with aqueous 60% CH,CN (150 ml), and lyophilized to yellow solids. physiological target sequences.Complications insubstrate Enzyme Digests-Peptide mixtures were dissolved by warming in design arise when there are several target sequences which dimethyl sulfoxide. Insoluble material was pelleted by centrifugation. are cleaved by a single enzyme. A relevant example is the Concentrations of Dnp-containingpeptides were determined by = 16,000 M" cm"). HIV' protease, which processes a wide variety of differing measuringabsorbance at 375 nm(Dnp Substrate mixtures were diluted to 250 p M final concentration in proproteins essential for the maturation of HIV (1). The essential feature of optimal substrate design is rapid assay buffer (200 mM NaC1, 50 mM Tris, 5 mM CaC12,0.05% Brij, pH 7.6). Standard substrate,Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH,, turnover (enhanced kcat/&) of target peptides. To minimize was added to theD and miscellaneous amino acid mixtures to a final the timerequired forthe development of an optimal substrate, concentration of 15 p ~ The . digests were initiated by adding recoma method hasbeen developed which allows the rapid mapping binant human fibroblast collagenase or collagenase/EDTA (control) to each mixture(250 p ~ 10-15 , p~ per substrate). The reactions were * The costs of publication of this article were defrayed in part by followed by monitoringthe increases intryptophan fluorescence (excitation, 280/emission,346) of themixtures,andthey were the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 quenched with EDTA after 30-50% hydrolysis of Dnp-Pro-Leu-GlyLeu-Trp-Ala-D-Arg-NH,. solely to indicate thisfact. HPLCICF-FAB-MS-Chromatography was performed usinga The abbreviationsused are: HIV, humanimmunodeficiency virus; HPLC, high pressure liquid chromatography; CF, continuous flow; Waters 600 solvent delivery system and two Waters Delta Pak CIR FAB-MS, fast atom bombardment mass spectrometry; Boc, t-butox- columns connected in series(150 X 3.9 mm, inner diameter). Solvent ycarbonyl; Bzl, benzyl; Chx, cyclohexyl; Dnp, dinitrophenyl; Z, ben- A consisted of aqueous 0.1% trifluoroacetic acid with 1% glycerol, and solvent B consisted of aqueous 0.1% trifluoroacetic acid, 1% zyloxycarbonyl; Tos, tosyl; Om, ornithine. ~~

1434

Mapping

Substrate

1435

glycerol, and 60% CH3CN. Chromatograms were developed over 90 min using either linear or concave gradients. Flow rates for all analyses were 1 ml/min. The liquid chromatography effluent was split, and about 10 pl/min was directed into the probe of a VG 70s double focusing mass spectrometer. The magnetic field was scanned repetitively once every 5 s from m/z 500 to m/z 1500. The ion source temperature was maintained at 50 "C, and the accelerating voltage was 5 kV.Alternatively, the chromatography was as above except 1% thioglycerol was used in place of glycerol in both solvent A and B. The liquid chromatography effluent was split, and about 10 pl/min was directed into the frit-FAB probe of a JEOL SX-102 mass spectrometer. The magnetic field was scanned repetitively once every 6 s from m/z 100 to m/z 1000. The ion source temperature was maintained at 55 "C, and theaccelerating voltage was 10 kV. RESULTS AND DISCUSSION

The syntheses of substrate mixtures were accomplished using routine solid-phase peptide synthesis techniques except at positions substituted with equimolar mixtures of amino acids. At these mapping sites, the chemistry was modifiedand a 2-fold excess of resin was used. The use of the activated amino acid mixture as the limiting reagent produced an approximately equimolar ratio of the final peptides despite the different coupling rates of the various amino acids (8). Peptides were cleaved from the solid-phase support using anhydrous HF followed by exhaustive extraction with trifluoroacetic acid. The desired stoichiometry at themapping positions was verified by amino acid analysis (data notshown). Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. Minutes Each of these substrate stock mixtures was hydrolyzed with recombinant human fibroblast collagenase. The reactions FIG. 1. HPLC/CF-FAB-MStrace of L-amino acid Phubstiwere initiated by adding collagenase or collagenase/EDTA tuted heptapeptide mixture. Chromatography was performed (control) to each mixture and quenched with EDTA after 30- using the chromatography system described under "Experimental Procedures" using a linear gradient. Structure, UV trace at 375 nm, 50% hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Argand reconstructed ion chromatogram (RZC)are shown. NH2. Each digest was chromatographed by HPLC using reversed-phase separation conditions optimized for the particular substratemixture. Eluting peaks were integrated by peak area or peak height (Dnp t375= 16,000 M-' cm"). mlz Under the competitive conditions employed in these digests, the relative specificity for any two competing substrates ( i e . kcat/Kmvalue) is given by the ratios of the values of ln(S/So) for each substrate at the time of quenching (9). This kinetic analysis is general and can be applied to two or more substrates thatare competing for flux through the same enzyme regardless of substrate concentration. The presence of inhibi___i itors in a mixture will decrease the absolute rate of turnover Scan Number of all substrates but will not affect the substrate specificity, FIG. 2. Mass chromatograms for the liquid chromatograkcat/Km.Potent inhibitors, therefore, can be detected although the identity of the inhibitory peptide(s) will not be readily phy-mass spectrometry analysis of the mixture represented in Fig. 1 showing differentiation of Leu- and Ile-containing discernible. The important point is that substrate specificity peptides. Top, m/z 299 represents the w3 ion for the Leu-containing is determined by the ratios of &/Km alone (10). The inte- peptide; middle, m/z 313 represents the W Q ion ~ for the Ile-containing grated peak areas were used to calculate (In (S/So)) for each peptide; bottom, m/z 904 represents the protonated molecular ion for substrate from the digests uersus controls. Comparison of both peptides. Chromatography was as in Fig. 1 except 1%thioglycthese values to Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 as erol was used in place of glycerol in both top and middle. The liquid effluent was split, and about 10 pl/min was directed standard was used to translate relative kCat/Km to absolute chromatography into the frit-FAB probe of a JEOL SX-102 mass spectrometer. The values and allowed comparison of results across all six digests. magnetic field was scanned repetitively once every 6 s from m/z 100 The composition of each peak in the chromatogram was to m/z 1000, beginning approximately 60 min after the chromatodetermined by HPLC/CF-FAB-MS. The FAB spectrum of graphic injection. The ion source temperature was maintained at each synthetic peptide contained molecular weight informa- 55 "C, and the accelerating voltage was 10 kV. tion (in the form of (M + H)+) andstructurally informative fragmentation. Fig. 1, top, shows the HPLC trace (UV = 375 along with those of two sequence-specific ions m / z 299 and nm) of the L-amino acid Pi-substituted heptapeptide mixture. m / z 313 representing ions unique to theleucine and isoleucine A reconstructed total ion chromatogram ( m / z 800-1000) un- Pi-substituted peptides, respectively (11).This approach alder identical conditions is shown in Fig. 1, bottom. In favorable lowed assignment of most of the individual components in cases, fragmentation patterns allow differentiation of isobaric the complex mixtures of peptides without the necessity of compounds. As an example, Fig. 2 shows the mass ,chromat- tedious and time-consuming isolation procedures. Analysis of the results from the six enzyme digests gave ograms of the protonated parent ions ( m / z 904, Fig.2, bottom) 299

1W

120

140

360

180

200

220

c

240

1436

Mapping

Substrate

both expected and unexpected results. Incubations of the Damino acid Pi and Pi -substituted heptapeptide mixtures with collagenase and examination of the resulting hydrolysates by HPLC revealed little change in the chromatograms over the time required for 50% hydrolysis of Dnp-Pro-Leu-Gly-LeuTrp-Ala-D-Arg-NH, (data not shown). These data indicate that D-amino acids are not tolerated by collagenase at the Pi or Pi positions. A variety of structurally diverse L-amino acid replacements is well accommodated at the Pi site (Table I). The normal amino acid at P; in human skin a1 chain of collagen is Ala. However, the S i site of collagenase apparently is able to adjust to many naturally occurring L-amino acids with the notable exceptions of the acidic residues (Asp and Glu), the sterically mobile residue Gly, and the imino acid Pro. The fastest turnover was obtained with the substrate containing His at Pi, and the basic residue Arg also gave significant enhancement. The map containing miscellaneous amino acid replacements at PI showed a similar allowance of a wide variety of amino acids as seen with the naturally occurring Lamino acid mixture (Table I). These results are qualitatively consistent with other studies of the substrate specificity of the human collagenases. Sottrup-Jensen and Birkedal-Hansen (12) profiled the collagenase-mediated hydrolysis of a TABLE I Substrate turnover for various Pi-substituted peptides

panel of serum protease inhibitors. These authors showed that thehuman a,-macroglobulin bait region isreadily cleaved by collagenase at a Gly-Leu scissile bond with Arg at the P; position (12). Also, Netzel-Arnett et al. (13) recently reported astudy of alternative peptide substrates for collagenase. Again, the enzyme displayed latitude at P; with hydrophobic amino acids and arginine being well tolerated, whileGlu substitution led to a sharp reduction in turnover. Substrate mapping of Pi with naturally occurring L-amino acids did not identify any peaks indicative of substantially enhanced substrates versus Leu (Table 11).However, turnover of the miscellaneous amino acids substrate mixture at the P; position revealed several substrates with enhanced turnover. Inspection of Table I1 reveals a marked preference for medium sized nonbranched and uncharged hydrophobic amino acids as substrates. The peptide containing Cys(Me) at Pi was found to be the optimal replacement for Leu P:, while norvaline and norleucine showed modest increases in turnover. To verify that Cys(Me) at Pi andHisat P; enhance turnover, substrates incorporating these individual substituTABLE I1 Substrate turnover for various P;-substituted peptides Amino acid substitution at P;

Relative K m)

(W

L-Amino acids NT" LY s His NT" L-Amino acids NT" 1.4 Arg LYs 0.5 His Gln 3.2 Asn and Ser (co-elution) NT" 2.7 Arg Gly and Thr(co-elution) 0.4 Gln 1.3 NT" Asp and Glu (co-elution) Asn 1.4 0.2 1.4 Ser Ala 0.4 Pro 0.4 Asp and Gly and Glu (co-elution) 0.2 1.5 Thr Tyr NT" Val 1.1 Ala 0.8 Met NT" Pro 0.7 Ile 1.9 TYr LOb Leu 1.9 Val NT" Phe 2.3 Met NT" 1.5 Ile Trp Miscellaneous amino acids 1.1 Leu 0.2 Ornithine L o b Trp NT" (p-NH2)-Phe 1.0 Phe 0.2 Sar' or @-Alaand Aba+ isomer Miscellaneous amino acids (co-elution) 1.5 Ornithine and (p-NH2)Phe(co-elution) 0.9 Sar or @-Ala NT Sar' or @-Ala 0.7 Abad isomer NT Abad isomer NT" Abad isomer NT Sar or @-Ala NT" Abad isomer 0.8 8-Aminooctanoic acid 3.2 0.5 Abad isomer Cys(M4 1.5 Norvaline NT Abad isomer 0.3 8-Aminooctanoic acid 1.3 Abad isomer 1.1 Norleucine 1.3 Cys(Me) 0.8 1.2 Tyr(Me) Norvaline NT" (N-Me)Ile NT (N-Me)Ile and (N-Me)Leu(co-elution) NT" (N-Me)Leu 1.2 Norleucine 0.9 @-NO,)Phe 1.0 Tyr(Me) 0.4 homo-Phe or (N-Me)Phe 1.1 (p-NO,)-Phe 0.4 homo-Phe or (N-Me)Phe and Tyr(Et) 0.2 homo-Phe and (N-Me)Phe (co-elution) (co-elution) 0.8 Tyr(Et) 0.5 (p-C1)Phe 0.6 (p-Cl)Phe NT" Cyclohexylalanine 1.1 Cvclohexvlalanine NT, no turnover; indicates that the ratewas less than 10%of the NT, no turnover; indicatesthat the ratewas less than 10%of the internal standard. internal standard. All k,,,/K, relative to Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg- All kJKm relative to Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-ArgNH,. NH,. e Sar, sarcosine. Sar, sarcosine. Aba isomers used are aminobutyric acid, aminoisobutyric acid/ Aba isomers used are aminobutyric acid, aminoisobutyric acid/ (N-Me)Ala, andy-aminobutyric acid. (N-Me)Ala, andy-aminobutyric acid. Amino acid substitution at Pi

Relative ( kaJKm)

"

Substrate Mapping

1437

tions, Dnp-Pro-Leu-Gly-Cys(Me)-Trp-Ala-D-Arg-NH2 and obtained in terms of the substrate specificity ratio k,,,/K, Dnp-Pro-Leu-Gly-Leu-His-Ala-D-Arg-NH2, were synthesized indicate which peptides in the mixture merit further study. and tested in a competition assay with the standard substrate To assure that a given substitution is truly rate-enhancing it verify the structureof enhanced substrates by as above. The k a t / K mvalues of these individual substrates is important to were 2.9 and 4.8 relative to Dnp-Pro-Leu-Gly-Leu-Trp-Ala-direct chemical synthesis and enzymatic evaluation and to D-Arg-NH,, in reasonable agreement with the values derived confirm the identity of the cleavage site. from the initial digests. Cleavage of these modified compounds NoteAdded i n Proof-Recently, several reports have appeared at theGly-Leu bond was verified by co-elution of the product, describing uses of synthetic peptide libraries. Birkett et al. (Birkett, Dnp-Pro-Leu-Gly-OH, with a synthetic standard on reverse A. J., Soler, D. F., Wolz, R. L., Bond, J. S., Wiseman, J., Berman, J., phase HPLC analysis. In order to assess the effect of simul- and Harris, R. B. (1991) Anal. Biochem. 196, 137-143) detailed the taneous modification at Pi and PI, we prepared Dnp-Pro- use of small synthetic peptide protease substrates and sequencing to Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2. The double substi- elucidate protease substrate specificity. Flynn et al. (Flynn, G. C., tuted substrate had a k,,,/K, value of 9.7 relative to Dnp- Pohl, J., Flocco, M. T., and Rothman, J. E. (1991) Nature 3 5 3 , 7 2 6 Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 representing an order 730) synthesized a known length, undefined sequence library and studied the peptide binding specificity of the heat shock protein of magnitude increase in turnover. family. Lam et al. (Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, Genetic approaches have also been described that have the V. J., Kazmierski, W. M., and Knapp, R. J. (1991) Nature 354, 82capacity to generate larger numbers of peptide/protein com- 84) produced a “one-bead, one-peptide”library and reported screening et al. binations for screening libraries. Reidhaar-Olson and Sauer of a@-endorphin monoclonal antibody.Finally,Houghten (14)utilized the technique of combinatorial cassette mutagen- (Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, T., and Cuervo, J. H. (1991) Nature 3 5 4 , 84-86) described an esis to map substitutions that allowed retention of functional C. iterative approach utilizing synthetic peptide combinatorial libraries repressor activity. Recently there have been reports describing t o screen for monoclonal antibody binding or antimicrobial activity. the utility of epitope libraries constructed from phage fUSE5 (15) and random peptide libraries from coliphage M13 (16). Unfortunately, the genetic approaches are only able to idenREFERENCES tify proteins containingthe naturally occurring L-amino acids. 1. Henderson, L. M., Copeland, T. D., Sowder, R. C., Schultz, A. The concept of screening peptide mixtures for a specific M., and Oroszlan, S. (1988) in Human Retroviruses, Cancer, activity was originally described in the mimotope mapping and Aids: Approaches to Preuention and Therapy (Bolognesi, D., ed) pp. 135-147, Alan R. Liss, Inc., New York technique of Geysen et al. (17). Standard solid-phase peptide 2. Caprioli, R. M. (1990) Anal. Chem. 62,477A-485A synthetic techniques have been used to assemble mixtures of 3. Caprioli, R. M., Fan, T., and Cottrell, J. S. (1986) Anal. Chem. peptides (18).These chemical approaches permit the incor58,2949-2954 poration of unnatural amino acids allowing the preparation 4. Ito, Y., Takeuchi, T.,Ishii, D., and Goto, M. (1985) J . Chromatogr. of unique syntheticmixtures of proteasesubstrates. The 346,161-166 5. Stack, M. S., and Gray, R. D. (1989) J. Biol. Chem. 2 6 4 , 4277power of this optimization method lies in the combinatorial 4281 capacity for individual site mapping. The only perceived limG. B., Van Wart, H. E., and Birkedal-Hansen, H. (1987) itation of this technique is the inability to assign distinct kcat 6. Fields, J. Biol. Chem. 262,6221-6226 and K,,, values to individual peptides. However, the method 7. Masui, Y., Takemoto, T., Sakakibara,S., Hori, H., and Nagai, Y. offers the significant advantage that relative rates for a large (1977) Biochem. Med. 17,215-221 8. Rutter, W. J., and Santi, D. V. (November 16, 1989) U. S. Patent number of substrates can be determined with a single measWO 89/10931 urement. Incorporation of optimal substitutions can quickly Abeles, R. H., Frisell, W. R., and Mackenzie, C. G. (1960) J. Biol. 9. lead to order of magnitude enhancements of substrate turnChem. 2 3 5 , 853-856; Correction (1960) J. Biol. Chem. 2 3 5 , over as seen above. no. 6 A detailed knowledge of the subsite specificity of collagen- 10. Fersht, A. (1985) in Enzyme Structure and Mechanism, (Fersht, A., ed) p. 111-112, W. H. Freeman and Co., New York ase is important for the development of synthetic substrates and inhibitors. Modifications which increase specificity ( i e . 11. Johnson, R. S., Martin, S. A., Biemann, K., Stults, J. T., and Watson, J. T. (1987) Anal. Chem. 59,2621-2625 kCat/K,)may translate into corresponding increases in inhib- 12. Sottrup-Jensen, L., and Birkedal-Hansen, H. (1989) J. Biol. itor potency (19-22). The results above represent a systematic Chem. 264,393-401 approach to studying this specificity. Inthe example de- 13. Netzel-Arnett, S., Fields, G. B., Birkedal-Hansen, H., and Van Wart, H. E. (1991) J. Biol. Chem. 266,6747-6755 scribed, more than 60 amino acid substitutions were quickly surveyed at thePi and Pi positions, respectively. The results 14. Reidhaar-Olson, J. F., and Sauer, R. T. (1988) Science 2 4 1 , 5357 suggest a significant latitude exists at Pi with >20 natural 15. Scott, J. M., and Smith, G. P. (1990) Science 2 4 9 , 386-390 and unnatural amino acids giving rate enhancement over 16. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990) Science tryptophan (Table I). However, the Pi position has a rather 249,404-406 narrow specificity with only 3 amino acids proving better than 17. Geysen,H. M., Rodda, S. J., and Mason, T. J. (1986) Mol. Immunol. 2 3 , 709-715 leucine (Table 11). F. S., Towery, D. S., Bulock, J. W., Whipple, D. E., Fok, In conclusion, the method described here is generally ap- 18. Tjoeng, K. F., Williams, M. H., Zupec, M. E., and Adams, S. P. (1990) plicable for the rapid optimization of any protease substrate Int. J. Pept. Protein Res. 3 5 , 141-146 k,.,/K, and offers a unique and efficient approach for analysis 19. Westerik, J. O., and Wolfenden, R. (1972) J . Biol. Chem. 247, 8195-8197 of substrate specificity. High performance liquid chromatography/continuous flow fast atom bombardment mass spec- 20. Thompson, R. C. (1973) Biochemistry 1 2 , 47-51 R. C., andBauer, C.-A. (1979) Biochemistry 18,1552trometry offers a rapidand reliable way of assigning structure 21. Thompson, 1.5.58 and analyzing substrate hydrolysis in complex mixtures with- 22. Barti&, P. A., and Marlowe, C. K. (1983) Biochemistry 2 2 , out having to first isolate the individual peptides. The results 4618-4624