THE JOURNAL OF BIOLOGICAL CHEMISTRY C C ’ 1987 by The American Society of Biological Chemists, Inc.

The Reactive Site of Human a2-Antiplasmin*

Vol. 262, No. 13, Issue of May 5, pp. 6055-6059, 1987 Printed in U.S.A.

(Received for publication, October 28, 1986)

Bie-Huoy ShiehS and James Travis@ From the $Department of Biochemistry, University of Georgia, Athens, Georgia 30602

Human a2-antiplasmin rapidly forms a stable, equi- molar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reac- tive site of the inhibitor with the concomitant release of a small peptide fragment which apparently repre- sents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of a2-antiplasmin encompasses a P1-P’, Arg-Met sequence. However, un- like a,-1-proteinase inhibitior which has a Met residue in the PI-position, oxidation of a2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mech- anistic importance of the P’l-residue during enzyme inactivation by this inhibitor.

The interaction of mammalian serine proteinases with their controlling plasma inhibitors has been suggested to involve rapid association of the two proteins and only very slow or negligible dissociation (1). Such studies have also indicated that at least five of these inhibitors, al-proteinase inhibitor (al-PI)‘, al-antichymotrypsin, antithrombin 111, Ci-inhibitor (Ci-Inh), and a2-antiplasmin (a2-AP) are related and, there- fore, members of a super family of inhibitors, referred to as serpins (2) because of homologies in both primary structure and mechanism of action. Indeed, all five inhibitors are unique in that they form stable, equimolar complexes with serine proteinases which cannot readily be dissociated by either acidification or addition of denaturing agents (1).

The mechanism by which proteinase inhibition occurs is not clear, but it apparently involves the interaction of the a- carbonyl group of the reactive site PI-residue of the inhibitor (according to the notation of Schechter and Berger (3)) and the a-hydroxyl group of the reactive seryl residue of the proteinase. Whether the complex formed is of the Michaelis type or involves tetrahedral or acyl intermediates remains to be established; however, it is known that complex dissociation can be attained by incubation with various nucleophilic agents

* This research was supported in part by grants from the National Lung Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§To whom correspondence should be addressed. ’ The abbreviations used are: al-PI, a,-proteinase inhibitor; a2-AP,

a,-antiplasmin; Ci-Inh, Ci-inhibitor; H-D-Val-Leu-Lys-paranitroan- ilide, H-D-valyl-leucyl-lysyl-paranitroanilide; Cbz-Ile-Glu-(-OR)- Gly-Arg-paranitroanilide, carbobenzoxyl-isoleucyl-glutamyl-orni- thyl-glycyl-arginyl-paranitroanilide; DFP, di-isopropylfluorophos- phate.

(4, 5 ) . This not only results in the release of free enzyme but also in cleavage of the inhibitor into two fragments by solvo- lysis at its reactive site, making it relatively simple to delin- eate the peptide bond attacked during proteinase inhibition.

Using such an approach four of the five predominant ser- pins in plasma have been extensively investigated (5-8), a2-

AP being the exception. This latter inhibitor is a single-chain glycoprotein with M , near 70,000 whose concentration in plasma is approximately 0.9 pM (9, 10). Its major functions are to control the activity of plasmin and trypsin (1, 11) by rapidly forming stable 1:l equimolar complexes, and it has been shown that during the interaction with plasmin the inhibitor is linked to the light chain of the enzyme which also contains the reactive site serine residue (4). Examination of both the peptide and the postcomplex form of a,-AP released after complex dissolution has suggested that its reactive site encompasses a Leu-Met peptide bond (4). However, the leucyl residue in the Pl position is unexpected considering the spec- ificity of both plasmin and trypsin for cleavage after basic amino acid residues. Furthermore, it was deduced by use of the enzyme carboxypeptidase Y, which is known to give abnormally high leucine release through a combination of endopeptidase cleavage after this residue in proteins and exopeptidase release of the free amino acid (12).

Recently (13), the nucleotide sequence of a cDNA clone coding for the carboxyl-terminal 274-amino acid residues of human a2-AP was reported. These data indicated that the reactive site encompasses a Met-Ser peptide bond, although it was also reported that on the basis of previous results (4) an Arg-Met sequence was possible. The latter has also been suggested, separately (14), but without proof. In this report we confirm the carboxyl-terminal sequence representing the P’l region of a2-AP (4). We also show data giving an overlap- ping sequence which allows us to conclude that the reactive site of a2-AP does, indeed, encompass an Arg-Met peptide bond. Finally, we provide experimental evidence to indicate that a2-AP is also a primary inhibitor of trypsin and that the methionyl residue in the P’l position is of lesser importance since oxidation fails to change the association rate with either this enzyme, plasmin, or chymotrypsin.

EXPERIMENTAL PROCEDURES

Materials Outdated human plasma was obtained from the American Red

Cross, Atlanta, GA. Porcine trypsin, bovine a-chymotrypsin, and DFP were from the Sigma. The V8 serine proteinase from Staphylo- coccus aureus was a gift of Dr. J. Potempa of this department. Human neutrophil elastase was purified by the method of Baugh and Travis (15). Human plasmin was purchased from Boehringer Mannheim. The plasmin substrate H-D-Val-Leu-Lys-paranitroanilide and the trypsin substrate Cbz-Ile-Glu-(-OR)-Gly-Arg-paranitroanilide were from Kabi (Stockholm). The bovine chymotrypsin substrate Suc-Ala- Ala-Pro-Phe-paranitroanilide was a gift of Dr. James Powers, Georgia Institute of Technology, Atlanta, GA.

6055

6056 The Reactive Site of Human az-Antiplasmin Methods

Purification of aZ-Antiplasmin-Human az-AP was isolated from outdated plasma by the procedure of Wiman (16). A final purification step involved preparative electrophoresis according to Pajdax (17). The inhibitor was stored in 0.04 M sodium phosphate buffer, pH 7.0.

Active Site Titrations-Active site titration of porcine pancreatic trypsin was performed by the method of Chase and Shaw (18).The enzyme was then used to determine the activity of az-AP (16) and the latter for active site titration of human plasmin. Bovine a- chymotrypsin and human neutrophil elastase were titrated against e previously standardized preparation of human a,-PI (19).

Sequence Analysis-Amino-terminal sequence analyses of 02-AP, porcine trypsin, and az-AP. trypsin complexes were performed using either a Beckman model 890C Protein Sequencer or an Applied Biosystems 470A gas-phase system, using programs designed by the manufacturers. Samples were dissolved in 0.2 M ammonium bicar- bonate buffer, pH 7.9, prior to analysis. Phenylthiohydantoin deriv- atives were identified by isocratic separation (7) or with an on-line analyzer (Applied Biosystems).

Formation of az-Antiplasmin Complexes with Trypsin-One hundred nanomoles of az-AP and 80 nmol of trypsin, both in 0.2 M NH,COs buffer, pH 8.0, were incubated together for 1 min a t room temperature. The sample was then transferred to the sequenator and dried under vacuum before initiation of the program. Controls con- taining trypsin inactivated with DFP, aZ-AP, or equimolar mixtures of each, were also subjected to sequence analysis.

Limited Proteolysis of a2-Antiplasmin-Human az-AP was modi- fied by limited proteolysis with either human neutrophil elastase or S. aureus V8 serine proteinase. Inhibitor (100 nmol) and V8 protein- ase (8.5 nmol) were incubated together in 0.2 M NH,CO, buffer, pH 8.0, at 37 "C, and loss of inhibitory activity was monitored over a 28- h period. Inactivation of oz-AP (100 nmol) by neutrophil elastase (1.5 nmol), using the same buffer system, was more rapid and was nearly complete (80%) in 40 min. At the end of the given time periods, DFP was added for proteinase inactivation and the mixtures applied to a preparative gel electrophoresis system using a linear gradient from 7-12.5s acrylamide (20). Individual peptides were then eluted as described elsewhere (7) and subjected to amino-terminal sequence analysis using the gas-phase protein sequenator.

Measurement of Association Rates-The second-order association rates for human az-AP (both native and oxidized) with human plas- min, porcine trypsin, and bovine chymotrypsin were determined by the method of Bieth (21). Equimolar mixtures of enzyme and inhibitor (based on the active site-titrated activities of each protein) were incubated for increasing time periods a t 25 "C in a total volume of 900 p10.1 M Tris-Hcl, pH 7.5, and residual enzyme activity measured by the addition of saturating amounts of specific proteinase sub- strates. The final enzyme concentrations were as follows: plasmin (79 nM), trypsin (16.5 nM), and chymotrypsin (5.2 nM).

Oxidation of a2-Antiplasmin-az-AP was incubated with either a 30 or 60-fold molar excess of SucNCl a t room temperature for 1 h in 0.1 M Tris-HC1, pH 8.8. The sample was dialyzed versus 0.04 M sodium phosphate, pH 7.4, to remove excess reagent, and residual inhibitory activity was measured against either plasmin or trypsin. Control samples were treated in the same manner but without the addition of SucNCl. Samples of al-PI were also treated with SucNCl in order to insure that oxidation was occurring, as measured by loss of inhibitory activity toward porcine pancreatic elastase (19).

Methionine Analysis-The conversion of methionine to methionine sulfoxide residues in az-AP by SucNCl oxidation was measured ac- cording to the procedure of Schechter et al. (22). The modified protein was subjected to CNBr cleavage (150-fold molar excess for 18 h) in 70% formic acid, in order to convert all unoxidized methionine residues. After lyophilization the sample was subjected to hydrolysis in 1.0 ml of 6 N HC1, containing 1.7 mg of dithioerythritol, for 24 h a t 100 "C, in vacuo, in order to reduce methionine sulfoxide residues back to free methionine. A white precipitate arising from the dithio- erythritol was removed by Millipore filtration, and the sample was analyzed on a Beckman model 119C1 amino acid analyzer. The value obtained for methionine was taken as the quantity oxidized by action of SucNCl on the native protein after subtraction for uncleaved methionyl residues obtained in control samples containing only native aZ-AP.

RESULTS

Properties of Human az-Antiplasmin-As purified, human a2-AP migrated as a single band after sodium dodecyl sulfate-

gel electrophoresis (Fig. 1A) with M , near 70,000. The homo- geneity of the final product was confirmed by amino-terminal sequence analysis (11 cycles) which yielded only a single residue at each step. The sequence, NHz-Asn-Gln-Glu-Gln- Val-Ser-Pro-Leu-Thr-Leu-Gly-Lys was found to be identical through the first several residues to that originally reported by Wiman and Collen (10,16). The product was stable in 0.04 M sodium phosphate buffer, pH 7.4, for several months a t 4 "C. Active site titration with a standardized preparation of porcine trypsin indicated that the protein contained 0.9 mol of active inhibitor/mol of protein, and this value was routinely utilized in determining E/I molar ratio.

Peptide Bond Cleavage in the az-Antiplasmin-Trypsin Com- plex-Sequence analysis of preformed complexes of az-AP and trypsin indicated the presence of three amino acid se- quences, two of which could readily be interpreted as repre- senting the amino-terminal sequences of the native inhibitor and enzyme, respectively (Table I). The third sequence is presumed to be derived from the interaction of the two pro- teins with each other, as incubation of DFP-inactivatd trypsin

C - 0

..X , 1-94 -68

I-45 '-29 -21

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis of native and neutrophil elastase modified a2-anti- plasmin. The gel channels contain: A, native az-AP; B, az-AP after 45-min incubation with neutrophil elastase (I/E ratio = 150:l); C, purified peptide released during inhibitor modification; D, M, stand- ards.

TABLE I Amino acid sequence of awmtiDlasmin-trvDsin comolex comDonents

C Y P l P Residues identified

1 Asn, Ile Asn, Ile, Met Met 2 Gln, Val Gln, Val, Ser 3

Ser Glu, Gly Glu, Gly, Val

4 Val

Gln, Gly Gln, Gly, Ser 5

Ser Val, Tyr Val, Tyr, Ser

6 Ser

Ser, Thr Ser, Thr, Phe Phe Trypsin (80 nmol) or trypsin previously inactivated by treatment

with DFP, was mixed with 100 nmol of az-AP in 0.2 M NH4C03 buffer, pH 8.0. Since the activity of the inhibitor was approximately 90% and the inhibitor 8095, the molar ratio of active trypsin to inhibitor was approximately 0.81.0. After 1 min at room temperature, the mixtures were applied directly to the sequenator for six cycles each. Amino acid residues were identified by high pressure liquid chromatography. Because of the presence of multiple sequences as well as the individual ones identified, repetitive yields could not be determined.

The Reactive Site of H u m a n cu2-Antiplasmin 6057

with a2-AP only yielded data corresponding to the amino- terminal structure of the two proteins. Furthermore, analysis of active trypsin did not give any other unusual sequences, indicating that the new sequence had not arisen because of autolysis of this proteinase during the incubation with inhib- itor. Therefore, it can be concluded that the new sequence, beginning NH2-Met-Ser-Leu-Ser-Ser-Phe-Ser-Val-Asn-, arises because of cleavage of the inhibitor at an X-Met bond, as originally observed by Wiman and Collen (10,16). Whether this is a direct result of complex formation or because of complex dissociation due to the high pH required for protein coupling to phenylisothiocyanate is, as yet, unclear.

Effect of Limited Proteolysis on a2-Antiplasmin-The in- cubation of native a2-AP with either s. aureus V-8 proteinase or human neutrophil elastase resulted in a rapid loss of inhibitory activity (Fig. 2). This was accompanied by peptide bond'cleavage (shown in Fig. 1 for the incubation with neu- trophil elastase) which yielded a modified form of the inhibitor ( M , = 58,000) (Fig. 1B) and a small peptide fragment ( M , near 1500) (Fig. 1, B and C). In each case, the latter was isolated by preparative gel electrophoresis and subjected to amino-terminal sequence analysis. As shown in Table 11, both gave peptide sequences which overlapped with that obtained during complex formation and/or dissociation, as described above, indicating that the Pll to P',+ sequence of a2-AP is Ala-Ala-Ala-Ala-Thr-Ser-Ile-Ala-Met-Ser-Arg-Met-Ser-Leu- Ser-Ser-Phe-Ser-Val-Asp-Arg-Pro-Phe-Leu-Phe, with an Arg-Met bond representing the reactive site P1-P', positions.

Effect of Oxidation on Association Rates of az-Antiplasmin- Because of the presence of a methionine residue at the P', position of a2-AP, experiments were performed to determine whether oxidation of this inhibitor would have any effect on its second-order association rate constants with other protein- ases. As shown in Table 111, trypsin was inactivated far more rapidly than either plasmin or chymotrypsin by a2-AP, with oxidation having essentially no effect on association rates, despite the fact that amino acid analysis had indicated that 2 of the 10 methionine residues of this protein were oxidized.

DISCUSSION

Previous data from this and other laboratories (1,5-8) have indicated that the amino acid representing the P1 residue in individual members of the serpin superfamily of inhibitors dictates primary specificity. Thus, enzymes targeted for con- trol by a given inhibitor must be able to cleave after the P, residue, as determined from either protein or synthetic pep- tide substrate hydrolysis (23). Indeed, even in members of the serpin family in which the protein had apparently lost its

: HLE

: I 1

TINE (mln) TIME (hrs)

FIG. 2. Inactivation of human az-antiplasmin by neutrophil elastase and S. uureus V8 proteinase. Inhibitor and enzyme were mixed as described in the text and aliquots taken at given time periods and assayed for residual trypsin inhibitory activity. a, neutro- phil elastase (HLE) and aZ-AP; b, V8 proteinase (V-8) and a,-AP.

TABLE I1 Sequence analysis of peptides released by limited proteolysis of

a2-antiplasmin with either neutrophil elastase or S. aureus V8 oroteinase

Cycle number

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

Residue identified"

Elastase-generated peptide

V-8 protease-gen- erated peptide

Met Ala Ser Ala Arg Ala Met Ala Ser Thr Leu Ser Ser Ile Leu Ala Phe Met Ser Val

Ser A%

ASP Met '4% Ser Pro Leu Phe Ser Leu Phe

"Amino acid residues were identified as described in the text. A repetitive yield of 94% was calculated for the elastase-generated peptide (Leu-6 to Leu-8) and 93% for the V8-generated peptide (Ala- 1 to Ala-8).

TABLE 111 Second-order association rate constants for a,-antiplasmin with

various Droteinases (M"s"J

Enzyme Native Oxidized antiplasmin antiplasmin"

Human plasmin 7.0 X 105 7.0 x 105 Bovine chymotrypsin 6.7 X 105 6.4 x 105 Porcine trypsin 1.3 X 107 1.1 x 107

N-chlorosuccinimide-treated.

inhibitory activity, e.g. ovalbumin, peptide bond hydrolysis has still been shown to occur at the remnant reactive site by the putative target proteinase (24). Therefore, the early sug- gestion (4) that a leucyl residue represents the P, amino acid in a2-AP seemed to be an exception to the general rule since, on the basis of the inhibitory specificity of this inhibitor, one would have expected a basic residue in this position. However, structural analysis of a cDNA clone coding for the carboxyl- terminal half of az-AP by Sumi et al. (13) has clearly estab- lished that a Leu-Met reactive site sequence is not possible. Instead, these results indicate either a Met-Ser reactive site on the basis of structural homology with other serpins or, alternatively, an Arg-Met if taken together with the sequence data of the carboxyl-terminal fragment formed after a,-AP. plasmin complex dissociation (4).

Our studies to determine the reactive site of n2-AP followed the procedures originally utilized for three other inhibitors, al-PI (5),Ci-Inh (7), and a,-antichymotrypsin (8). This in- volved (a) the forced cleavage of the reactive site peptide bond of the inhibitor through complex formation with a given proteinase; (b) sequence analysis of the modified inhibitor to determine the sequence from the P'l position on (Table I); and (c) enzymatic hydrolysis upstream from the reactive site to give a modified protein whose amino-terminal sequence overlapped with that beginning with the P'* residue (Table 11). This latter procedure always resulted in inhibitor inacti- vation and is presumed to be due to major conformational changes in the cleaved inhibitor, as originally shown for aI - PI (25). Indeed, it has been suggested that the inhibitory activity of individual serpins may be mediated by proteolytic

6058 The Reactive Site of Human as-Antiplasmin

attack at or near the reactive site by proteinases not targeted for inhibition (1). As shown in Table IV, there are several positions upstream from the reactive site region of the serpins which are not only highly conserved but also potentially sensitive to inhibitor inactivation following cleavage after either glutamyl or alanyl residues, in agreement with the data obtained both here and previously (5,7,8). However, whether this is truly a common mechanism for inhibitor control re- mains to be established.

For az-AP we first chose to use neutrophil elastase to obtain an overlapping sequence, as this had originally been shown to rapidly inactivate this inhibitor (26). However, we tested several other enzymes and found that the V-8 proteinase also worked well to inactivate a2-AP. In both cases we were able to obtain peptide fragments whose sequences not only over- lapped with that obtained by cleavage of the trypsin-a2-AP complex but also with each other (Table 11).

The presence of an arginyl residue in the PI position of a2- AP supports the hypothesis that, at least in the serpin family, a major role is played by the amino acid in this position in determining inhibitor specificity. Indeed, even in the case of heparin cofactor I1 which has a leucyl residue in the Pl- position (27), yet is a potent inhibitor of thrombin when heparin is present (28), the specificity rule still holds since the inhibitor can act effectively as a chymotrypsin inhibitor in either the presence or absence of heparin (29). This would suggest that heparin can bind to thrombin in such a manner as to dramatically increase its interaction with HCII (29). Just how this change would allow strong binding for an arginine-specific proteinase to a leucyl residue in the Pl- position is, however, unknown and would require a detailed examination of a heparin-thrombin-heparin cofactor I1 crys- tal complex. Nevertheless, it does strongly infer that several other residues besides that in the P1 position contribute in order for inhibition to be of physiological importance, and this has been confirmed in studies on the specificity of other members of the serpin family. For example, both a2-AP and Ci-Inh have Pl-arginine residues (7). However, despite the fact that their general specificities are toward trypsin-like

enzymes neither is an effective inhibitor of the others target enzyme, i.e. az-AP does not inhibit plasma kallikrein (1) and Ci-Inh only very slowly forms complexes with either trypsin or plasmin (7).

One possible reason for the rather narrow specificity of the members of the serpin family may be due to sequence varia- bility near the reactive site which, with the exception of P’l, is exceedingly high in the region from P, to P’, (Table IV). It has recently been shown that in the ovomucoid family a high degree of variability occurs in areas which are in contact with the proteinase being inhibited, particularly in the region of the reactive site peptide bond (30), although insertions and deletions in these regions are strictly forbidden. Clearly, such changes have been made in order to increase both the rate of complex formation and the stability of the E.1 complex formed. From the data given in Table IV which indicates not only variability but also gaps and insertions near the reactive site, it seems likely that this will also hold true for the serpins. For this reason caution should be taken in attempting to establish reactive site sequences strictly on the basis of se- quence homology (13).

Surprisingly, we could not confirm the rapid reaction rates originally observed for the interaction of az-AP with human plasmin (1 I), the second-order association rate constants obtained in this report being some 50 times slower. We have no explanation for the differences between our data and those of others (ll), except for the fact that we worked with equi- molar mixtures of enzyme and inhibitor while the original studies were done in the presence of excess inhibitor. Actually, from the data given in Table I11 it would appear that a-,-AP might be an important controlling inhibitor of trypsin in all tissues, with the exception of blood where a,-M maintains control (I) , since its association rates, at least with the porcine enzyme, are far higher than those observed with other serpins.

The role of the P’, residue in the serpins has never been clearly established. In six of the members of this family it has involved either serine or threonine, suggesting a potential role for the hydroxyl group in this position. In fact, it has recently been shown that a mutant form of antithrombin 111, in which

TABLE IV Reactive site sequences of the serpin superfamily

Boxed residues were chosen on the basis of 50% or more homology among the 12 serpins listed. Homologies upstream and downstream from the reactive site are obvious, but residues close to the reactive site peptide bond differ greatly between proteins. The placement of gaps has been arbitrarily chosen and is required due to the different lengths of the putative reactive site loops. hLS2 (35) and HCII (40) are apparently identical serpins identified by homologies of partial cDNA clones coding for HCII. Standard one-letter amino acid abbreviations are used. Additional abbreviations used are: al-Achy, al-antichymotrypsin; AT 111, antithrombin 111; P-PAI, p- plasminogen activator inhibitor; HCII, heparin cofactor 11; hLS2, human leuserpin; bp2, barley protein 2; Ang, angiotensinogen.

Protein P,-Pl References

CYZ-AP a,-PI al-Achy Contrapsin AT I11 @-PA1 CTInh HCII (hLS2) bPZ Ovalbumin Ang (rat) Ang (human)

~~~ ____

V T L ~ P F L F]

The Reactive Site of Human a2-Antiplasmin 6059

the p f1 serine has apparently been replaced by leucine, is 12. Christensen, u., and Clemmensen, I. (1977) Biochem. J. 163, inactive as an inhibitor of thrombin (31), supporting the 389-391 contention of a major role for this residue. In a2-AP, however, 13. Sumi, Y., Nakamura, Y., Aoki, N., Sakai, M., and Muramatsu,

the P’I residue is methionine and, although it may function 14. Linjen, H. R., Van Hoef, B., Wiman, B., and Collen, D. (1985) in a manner analogous to that of either threonine or serine, Thromb. Res. 3 9 , 625-630 it is surprising that oxidation does not have any effect on the 15. Baugh, R. J., and Travis, J. (1978) Biochemistry 15,836-841 function of this protein. This is in stark contrast to that of 16. Wiman, B. (1981) Methods Enzymol. 80.395-408

M. (1986) J. Biochem. (Tokyo) 100, 1399-1402

@,-PI where a methionine residue is in the apparently more 17. Pajdak, w. (1g73) Clin. Chim. Acta 4 8 9 113-115

important position (5) and to the @-plasminogen activator 19. Beatty, K., Bieth, J., and Travis, J, (1980) J , Bioi, Chem, 255 , 18. Chase, T., Jr., and Shaw, E. (1970) Methods Enzymol. 19,20-27

inhibitor (32) which also has recently been shown to have an 3931-3934 Arg-Met sequence in the PI-P’I reactive site sequence. In 20. Barrett, A. J., Brown, M. A,, and Sayers, C. A. (1979) Biochem. both cases oxidation has a dramatic effect on inhibitory J. 181,401-418 activity, drastically reducing the rate of interaction with target 21. Bieth, J. G. (1980) Bull. Eur. Physiopathol. Respir. 16, (Suppl.)

enzymes. 183-195 If oxidation is not important in regulating the activity of 22. Schechter, Y., Burstein, Y., and Patchornik, A. (1975) Biochem-

@z-AP, then what is the reason for Positioning a methionine 23. Nakajima, K., Powers, J. c., Ashe, B. M., and zirnmerman, M. residue in the P’l-position? Clearly, it is not for the modula- (1979) J. Biol. Chem. 254 , 4027-4032 tion of inhibitory activity through enzymatic inactivation 24. Wright, H. T. (1984) J. Biol. Chem. 259, 14335-14336 since neutrophil elastase, which can cleave after methionyl 25. Loebermann, J., Tokuoka, R., Deisenhofer, J., and Huber, R. residues (23), prefers to hydrolyze between an alanine residue (1984) J. Mol. Biol. 177, 531-558

the inhibition Of which has been reported to 27. Griffith, M. J., Noyes, C. M., Tyndall, J. A,, and Church, F. C. be rapidly inactivated by a2-AP (ll), and is confirmed in this (1985) Biochemistry 2 4 , 6777-6782 report. Experiments are currently in progess to examine this 28. Tollefsen, D. M., Majerus, D. W., and Blank, M. K. (1982) J. feature of the inhibitor and to to determine whether it con- Biol. Chem. 257,2162-2169

chymotrypsin. Since the @-plasminogen activator inhibitor Acad. Sci. U. S. A. 8 2 , 6431-6434

also contains an Arg-Met sequence at the PI-PfI reactive site 30. Kato, I., Ardelt, W., Cook, J., Denton, A., Entie, M. W., Kohr,

(32) it would be interesting to determine whether this inhib- W. J., Park, F. J., Parks, K., Schatzley, B. L., Schoenberger, 0.

itor can also inactivate chymotrypsin. L., Tashiro, M., Vechot, G., Whatley, H. E., Wiecorek, A., Wiecorek, M., and Laskowski, M., Jr. (1987) Biochemistry 26 , 202-221

Acknowkdgments-we wish to thank Dr. John Wunderlich and 31. Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, Dr. Wieslaw Watorek for amino acid sequence analyses and Dr. Jan M. J., and Hathaway, W. E. (1986) J. Clin. Inuest. 7 7 , 887- Potempa and Dr. Guy Salvesen for helpful discussions during the 893 course of this work. 32. Ny, T., Sawdey, M., Lawrence, D., Millan, J. C., and Loskutoff,

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2. Carrell, R., and Travis, J. (1983) Trends Biochem. sci. 10,20-24 35. Ragg, H. (1986) Nu‘& id^ R ~ ~ , 14 , 1073-1088 3. Schechter, I., and Berger, A. (1967) BiOchem. Biophys. Res. h n - 36. Hejgaard, J., Rasmussen, S. K., Brandt, A., and Svenden, 1. (1985)

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