mapping the extended substrate binding site of cathepsin g and

7
THIS JOUPNAL OF Btomtxca Ca~vurrilr Vol. 2.54. No. 10, Isme of May 26, pp. 402?-4032, 1979 F?G#edin U.S.A. Mapping the Extended Substrate Binding Site of Cathepsin G and Human Leukocyte Elastase STUDIES WITH PEPTIDE SUBSTRATES RELATED TO THE al-PROTEASE INHIBITOR REACTIVE SITE* (Received for publication, September 14,1978, and in revised form, November 10,1978) Kiichiro Nakajima and James C. Powers+ From the School of Chemists, Georgia Institute of Technology, Atlantu, Georgia 30332 Bonnie M. Ashe and Morris Zimmerman From the Merck Znstitute of Therapeutic Research, Rahway, New Jersey 07065 The kinetic constants for the hydrolysis of a series of I-nitroanilide substrates by human leukocyte (HL) elastase and cathepsin G, porcine pancreatic elastase, and bovine chymotrypsin at pH 7.50 are reported. HL elastase and cathepsin G are currently thought to be the agents responsible for destruction of the lung in the disease emphysema. MeO-Sue-Ala-Ala-Pro-VaI-NA is an excellent substrate for HL elastase and is not hydro- lyzed by cathepsin G. The MeO-Sue-group increases the solubility of a substrate relative to the acetyl group. With HL elastase, this structural change increases the reactivity of the enzyme toward both I-nitroanilide substrates and chloromethyl ketone inhibitors. This indicates that HL elastase is interacting with at least 5 residues of a substrate (or inhibitor). Cathepsin G pre- fers Pb groups which are negatively charged such as Sue-, Suc(4F)-, Glt-, or Mal-. This enzyme, in common with many other serine proteases cannot accept a Pro residue at its Ss subsite. One of the better substrates for cathepsin G, Sue-Ala-Ala-Pro-Phe-NA, was not hy- drolyzed by HL elastase. These tools should be useful in the study of the biological function of HL elastase and cathepsin G. Two tetrapeptide 4-nitroanilide sub- strates related to the reactive site of the plasma (~1’ protease inhibitor (ai-antitrypsin) were studied. Both have a P1 Met residue and one, MeO-Suc-Ala-Ile-Pro- Met-NA, has the exact sequence of the Pa to Pt residues at the proteolysis site of al-PI (Johnson, D. A., and Travis, J. (1978) J. BioL Clrem. 253, 7142-7144). Both MeO-Sue-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-Ile- Pro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is in fact the best 4- nitroanilide substrate of cathepsin G yet reported. Ox- idation of MeO-Sue-Ala-Ala-Pro-Met-NA yielded two diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. In the preceding pa- per, Johnson and Travis (Johnson, D., and Travis, J. (1979) J. BioL Chem. 254, 4022-4026) show that oxida- tion of al-PI destroys its inhibitory activity. In concert, our results indicate that oxidation of the PI Met of (Ye- PI is capable to destroying its reactivity toward most serine proteases. Oxidation of al-PI by some component in cigarette smoke would offer one explanation in mo- * This investigation was supported in part by a grant from the Council for Tobacco Research. 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 USE. Section 1734 solely to indicate this fart. $ To whom correspondence may be addressed. lecular terms for the link between smoking and emphy- sema. Pulmonary emphysema is currently thought to result from proteolysis of the lung by proteases released from polymor- phonuclear leukocytes (Mittman, 1972; Turin0 et al., 1974; Hance and Crystal, 1975). Several enzymes have been isolated from the granule fraction of human leukocytes (Starkey, 1977). Two of these enzymes, HL’ elastase and cathepsin G, are probably responsible for the majority of tissue destruction in emphysema since both have been shown to attack lung elastin. In normal individuals the plasma protease inhibitor al- protease inhibitor (al-antitrypsin) inactivates any elastase or cathepsin G which is released from leukocytes. This inacti- vation involves formation of a stable complex between al-PI and the protease. Although details of the mechanism have not been worked out, complex formation involves formation of a sodium dodecyl sulfate-stable bond between the protease and the al-PI. Treatment of the complex under alkaline conditions yields a modfied al-PI which has a single new peptide bond cleavage (Johnson and Travis, 1976; Cohen et al., 1978). Modified LY,-PI with this same cleavage has been obtained after treatment of al-PI with proteases such as HL elastase, cathepsin G, and papain. This indicates that all proteases which are inhibitable by al-PI are reacting with a single reactive site. A tentative working hypothesis for the mecha- nism of inhibition of various proteases by al-PI would involve binding of the protease to the reactive site of CQ-PI with subsequent formation of a sodium dodecyl sulfate-stable link- age between the inhibitor and the enzyme. The sequence of the al-PI reactive site has recently been determined and consists of a Pq to P1’ Ala-Ile-Pro-Met*Ser(or Thr) sequence (cleavage occurs at the asterisk) (Johnson and Travis, 1978).’ A relatively few individuals with emphysema have a defi- ciency of al-PI. However, many patients with chronic obstruc- tive pulmonary disease have a history of smoking. Recently Janoff and Carp (1977) have demonstrated that smoke con- densate inactivates (ul-PI. In the adjacent paper, Johnson and 1 The abbreviations used are: HL, human leukocyte; Boc, t-butox- ycarbonyl; NA, 4-nitroanilide; SW, succinyl; Glt, glutaryl; Mal, mal- onyl; Suc(rlF), perfluorosuccinyl; MeO-Sue, methoxysuccinyl; HOBt, N-hydroxybenzotriazole; -ONp, 4nitrophenyl ester; Hepes. 4-(2-hv- droxyeth$)-I-piperazineethanesulfonic~acid~ PI, protea& inhibit& DMF, dimethvlformamide: THF, tetrahvdrofuran. The nomek&ture used for the individual amino acid residues (PI, P2, etc.) of a substrate and the subsites (S,, St, etc.) of the enzyme is that of Schechter and Berger (1967). 4027 by guest on February 4, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Mapping the Extended Substrate Binding Site of Cathepsin G and

THIS JOUPNAL OF Btomtxca Ca~vurrilr Vol. 2.54. No. 10, Isme of May 26, pp. 402?-4032, 1979 F?G#edin U.S.A.

Mapping the Extended Substrate Binding Site of Cathepsin G and Human Leukocyte Elastase STUDIES WITH PEPTIDE SUBSTRATES RELATED TO THE al-PROTEASE INHIBITOR REACTIVE SITE*

(Received for publication, September 14,1978, and in revised form, November 10,1978)

Kiichiro Nakajima and James C. Powers+ From the School of Chemists, Georgia Institute of Technology, Atlantu, Georgia 30332

Bonnie M. Ashe and Morris Zimmerman From the Merck Znstitute of Therapeutic Research, Rahway, New Jersey 07065

The kinetic constants for the hydrolysis of a series of I-nitroanilide substrates by human leukocyte (HL) elastase and cathepsin G, porcine pancreatic elastase, and bovine chymotrypsin at pH 7.50 are reported. HL elastase and cathepsin G are currently thought to be the agents responsible for destruction of the lung in the disease emphysema. MeO-Sue-Ala-Ala-Pro-VaI-NA is an excellent substrate for HL elastase and is not hydro- lyzed by cathepsin G. The MeO-Sue-group increases the solubility of a substrate relative to the acetyl group. With HL elastase, this structural change increases the reactivity of the enzyme toward both I-nitroanilide substrates and chloromethyl ketone inhibitors. This indicates that HL elastase is interacting with at least 5 residues of a substrate (or inhibitor). Cathepsin G pre- fers Pb groups which are negatively charged such as Sue-, Suc(4F)-, Glt-, or Mal-. This enzyme, in common with many other serine proteases cannot accept a Pro residue at its Ss subsite. One of the better substrates for cathepsin G, Sue-Ala-Ala-Pro-Phe-NA, was not hy- drolyzed by HL elastase. These tools should be useful in the study of the biological function of HL elastase and cathepsin G. Two tetrapeptide 4-nitroanilide sub- strates related to the reactive site of the plasma (~1’ protease inhibitor (ai-antitrypsin) were studied. Both have a P1 Met residue and one, MeO-Suc-Ala-Ile-Pro- Met-NA, has the exact sequence of the Pa to Pt residues at the proteolysis site of al-PI (Johnson, D. A., and Travis, J. (1978) J. BioL Clrem. 253, 7142-7144). Both MeO-Sue-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-Ile- Pro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is in fact the best 4- nitroanilide substrate of cathepsin G yet reported. Ox- idation of MeO-Sue-Ala-Ala-Pro-Met-NA yielded two diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. In the preceding pa- per, Johnson and Travis (Johnson, D., and Travis, J. (1979) J. BioL Chem. 254, 4022-4026) show that oxida- tion of al-PI destroys its inhibitory activity. In concert, our results indicate that oxidation of the PI Met of (Ye- PI is capable to destroying its reactivity toward most serine proteases. Oxidation of al-PI by some component in cigarette smoke would offer one explanation in mo-

* This investigation was supported in part by a grant from the Council for Tobacco Research. 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 USE. Section 1734 solely to indicate this fart.

$ To whom correspondence may be addressed.

lecular terms for the link between smoking and emphy- sema.

Pulmonary emphysema is currently thought to result from proteolysis of the lung by proteases released from polymor- phonuclear leukocytes (Mittman, 1972; Turin0 et al., 1974; Hance and Crystal, 1975). Several enzymes have been isolated from the granule fraction of human leukocytes (Starkey, 1977). Two of these enzymes, HL’ elastase and cathepsin G, are probably responsible for the majority of tissue destruction in emphysema since both have been shown to attack lung elastin.

In normal individuals the plasma protease inhibitor al- protease inhibitor (al-antitrypsin) inactivates any elastase or cathepsin G which is released from leukocytes. This inacti- vation involves formation of a stable complex between al-PI and the protease. Although details of the mechanism have not been worked out, complex formation involves formation of a sodium dodecyl sulfate-stable bond between the protease and the al-PI. Treatment of the complex under alkaline conditions yields a modfied al-PI which has a single new peptide bond cleavage (Johnson and Travis, 1976; Cohen et al., 1978).

Modified LY,-PI with this same cleavage has been obtained after treatment of al-PI with proteases such as HL elastase, cathepsin G, and papain. This indicates that all proteases which are inhibitable by al-PI are reacting with a single reactive site. A tentative working hypothesis for the mecha- nism of inhibition of various proteases by al-PI would involve binding of the protease to the reactive site of CQ-PI with subsequent formation of a sodium dodecyl sulfate-stable link- age between the inhibitor and the enzyme. The sequence of the al-PI reactive site has recently been determined and consists of a Pq to P1’ Ala-Ile-Pro-Met*Ser(or Thr) sequence (cleavage occurs at the asterisk) (Johnson and Travis, 1978).’

A relatively few individuals with emphysema have a defi- ciency of al-PI. However, many patients with chronic obstruc- tive pulmonary disease have a history of smoking. Recently Janoff and Carp (1977) have demonstrated that smoke con- densate inactivates (ul-PI. In the adjacent paper, Johnson and

1 The abbreviations used are: HL, human leukocyte; Boc, t-butox- ycarbonyl; NA, 4-nitroanilide; SW, succinyl; Glt, glutaryl; Mal, mal- onyl; Suc(rlF), perfluorosuccinyl; MeO-Sue, methoxysuccinyl; HOBt, N-hydroxybenzotriazole; -ONp, 4nitrophenyl ester; Hepes. 4-(2-hv- droxyeth$)-I-piperazineethanesulfonic~acid~ PI, protea& inhibit& DMF, dimethvlformamide: THF, tetrahvdrofuran.

’ The nomek&ture used for the individual amino acid residues (PI, P2, etc.) of a substrate and the subsites (S,, St, etc.) of the enzyme is that of Schechter and Berger (1967).

4027

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Page 2: Mapping the Extended Substrate Binding Site of Cathepsin G and

4028 Subsite Mapping of Cathepsin G and Leukocyte Elastase

Travis (1979) show that al-PI is inactivated by oxidation of methionine residues.

In this paper, we report the synthesis and kinetic studies with a number of 4-nitroanilide substrates for HL elastase and cathepsin G which provide information on the nature of the extended substrate binding site in these enzymes. Results with substrates related in sequence to the al-PI proteolysis site show that this sequence is reactive toward the two major serine proteases found in the granules of human leukocytes. In addition, oxidation at this site could account for the inac- tivation of al-PI upon treatment with oxidizing agents or smoke condensate.

EXPERIMENTAL PROCEDURES

Human leukocyte elastase and cathepsin G were supplied to us by Dr. James Travis of the University of Georgia. Porcine pancreatic elastase was purchased from Worthington. Chymotrypsin was pur- chased from Sigma. N,N’-Dicyclohexylcarbodiimide and N-hydroxy- benzotriazole (HOBt) were from Aldrich. Perfluorosuccinic anhydride was purchased from PCR Research Chemicals. Sue-(Ala)a-NA was generously given to us by Dr. Joseph Bieth, Universite Louis Pasteur, Strasbourg, France. The other 4.nitroanilides were synthesized in our laboratories.3

I-Nitroanilide Kinetics-The rates of hydrolysis of the 4-nitroan- ilides were measured bv adding 25 ~1 of the appropriate enzyme solution to 1.8 ml of a substrate solution in 0.1 M Hepes buffer at pH 7.50 containine 0.5 M NaCl and 10% dimethvlsulfoxide at 25°C. The increase in the absorbance at 410 nm was followed with a Beckman model 25 spectrophotometer. An c of 8800 at 410 nm was used (Erlanger et al., 1961). The kinetic constants were determined from the initial rates of hydrolysis by the Lineweaver-Burk method and are based on duplicate rate determinations at five separate substrate concentrations. Correlation coefficients were greater than 0.99.

The concentration of leukocyte elastase was determined by active site titration with Ac-Ala-Ala-NHN(CH3)CO-ONp (Powers and Gup- ton, 1977). The concentration of cathepsin G was determined with the Boc-TyrONp using the kinetic constants which are based on titrated enzyme (Powers et al., 1977).

RESULTS

The kinetic constants for the hydrolysis of several 4-nitroan- ilide substrates by human leukocyte and porcine pancreatic elastase are reported in Table I. The most effective substrate for HL elastase is MeO-Sue-Ala-Ala-Pro-Val-NA. This is an exact analog of the chloromethyl ketone elastase inhibitor, MeO-Sue-Ala-Ala-Pro-ValCHzCl, which is the most effective chloromethyl ketone inhibitor of HL elastase (Powers et al., 1977). As compared to an acetyl group, the methoxysuccinyl group gives a substrate (or inhibitor) increased solubility and, in the case of HL elastase, increased reactivity. The 4-nitroan- ilide with a P, Met in place of Val has a 400-fold lower k,.JK, value with HL elastase. However, MeO-Suc-Ala-Ile-Pro-Met- NA, which has the same sequence as the Pd to Pi residues of the o,-protease inhibitor reactive site, has a l&fold higher k&K, value than MeO-Sue-Ala-Ala-Pro-Met-NA.

The kinetic constants for the hydrolysis of Suc-Ala-Ala- Ala-NA have previously been measured with PP elastase (pH 8.0, 0.2 M Tris, 1% N-methylpyrrolidone, 25°C) and the elas- tase from pundent sputum (pH 8.0, 0.2 M Tris, 1 M NaCl, 25°C). The K,,,, kCat, and k,.JK,,, values were found to be, respectively, 2.4 II~M, 21.2 s-l, and 8.3 x 10” M-’ s-’ (Bieth et

’ Experimental procedures for the synthesis of most of the 4-ni- troanilides are presented in a miniprint supplement immediately following this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 78M-1642, cite author(s), and include a check or money order of $1.05 per set of photocopies.

al., 1974) and 1.8 mM, 0.45 s-‘, and 2.5 X 10” M’ so’ (Twumasi and Liener, 1977). The values we report for the PP elastase are very close to those reported by Bieth et al. (1974) and are accounted for by the slightly different pH values used in the two studies. In the case of the human elastases, the 4-fold discrepancy in kcat is probably due not only to a pH difference but also due to the fact that our kCat value is based on titrated enzyme.

The kinetic constants for the hydrolysis of a number of 4- nitroanilide substrates by human leukocyte cathepsin G are reported in Table II. Since very little data was available in the literature on the secondary substrate specificity of this enzyme, we began our search for a useful substrate by making Z-Gly-Leu-Phe-NA. This is a direct analog of the chloro- methyl ketone cathepsin G inhibitor Z-Gly-Leu-PheCH&l, which was the most effective of a considerable number sur- veyed (Powers et al., 1977). Unfortunately Z-Gly-Leu-Phe- NA proved to be insoluble in aqueous solutions even with the addition of organic solvents. Several soluble relatives, such as H-Gly-Leu-Phe-NA and Sue-Gly-Leu-Phe-NA, were tested, but all proved to be at least IO-fold less reactive than Suc- Ala-Ala-Phe-NA. We then studied tetrapeptide analogs of Ac- Ala-Ala-Pro-Phe-NA which was previously reported to be hydrolyzed more effectively by cathepsin G than the corre- sponding 4-nitroanilide with a P1 Leu residue (Zimmerman and Ashe, 1977). The tetrapeptide substrates with a P, nega- tively charged group (Mal-, Sue-, or Glt-Ala-Ala-Pro-Phe-NA) were more effective than those with no charge (AC- or MeO- Sue-) or those with a positive charge (HCl-H-Ala-Ala-Pro- Phe-NA or HBr.NH2(CH2)&O-Ala-Ala-Pro-Phe-NA). The tetrafluorosuccinylderivative (Suc(4F)-Ala-Ala-Pro-Phe-NA) was synthesized due to a report than certain trifluoroacetyl peptides are excellent inhibitors of HL elastase (Lestienne et al., 1977, 1978). Although the tetrafluoro compound was slightly more reactive with HL cathepsin G, the rate difference was not significant enough to justify its use rather than the more easily synthesized Sue-Ala-Ala-Pro-Phe-NA.

Cathepsin G will not tolerate a Pro residue in the Ps position of a substrate. Sue-Ala-Pro-Leu-Phe-NA was cleaved by the enzyme at the Leu-Phe bond with formation of Phe-NA. Thus, even though the enzyme prefers a PI Phe (compare Sue-Ala-Ala-Pro-Leu-NA with Sue-Ala-Ala-Pro-Phe-NA), the Pro residue prevents binding such that hydrolysis at the 4-nitroanilide bond could take place.

4-Nitroanilides with a PI Met or Met(O)-The two methi- onine nitroanilides were synthesized after it was learned that the Pi residue of the ai-protease inhibitor was a Met (Johnson and Travis, 1978). In fact the best cathepsin G substrate is MeO-Sue-Ala-Ala-Pro-Met-NA which has a 5-fold higher k,,J Km value than MeO-Sue-Ala-Ala-Pro-Phe-NA. Interestingly, the kcaL values for the hydrolysis of MeO-Suc-Ala-Ile-Pro- Met-NA by HL elastase, cathepsin G, and bovine chymotryp- sin were almost the same: 6.8, 4.5 and 2.8 s-‘, respectively. With MeO-Sue-Ala-Ala-Pro-Met-NA the kCat values show a 21-fold variation. Bovine trypsin did not hydrolyze either Met 4-nitroanilide.

Oxidation of MeO-Sue-Ala-Ala-Pro-Met-NA with Hz02 gave two diastereomeric sulfoxides. Both of these sulfoxides were inert at 2 mM concentration to HL elastase, cathepsin G, and bovine trypsin. However, they are hydrolyzed slowly by bovine chymotrypsin (Table III) with k&K, values of 400 to 700 lower than those of the unoxidized Met tetrapeptide. Neither Met(O) diastereomer was a competitive inhibitor of the HL elastase-catalyzed hydrolysis of MeO-Suc-Ala-Ala- Pro-Val-NA or of the cathepsin G catalyzed hydrolysis of Suc- Ala-Ala-Pro-Phe-NA.

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Subsite Mapping of Cathepsin G and Leukocyte Elastase 4029

TABLE I Kinetic constants for the hydrolysis of synthetic I-nitroanilide substrates by human leukocyte (HL) and porcine pancreatic (PP) elastase

Conditions were: pH 7.50,O.l M Hepes buffer, 0.5 M NaCl, 9.8% dimethylsulfoxide at 25°C. Substrate

EilZyiiW Substrate concentration K3”@ L kc., k<.,/Km

Ps PI Ps PY P, IPlM mM s-’ ),-L g-l

Sue- Ala- Ala- Ala-NA HL 0.4-7.6 3.7 2.1 570 PP 0.4-7.6 5.9 37. 6,300

Ac-Ala-Ala-Pro-Val-NA” HL 0.31 8.1 27,000 MeO-Suc- Ala-Ala-Pro-Val-NA HL 0.05-4.9 0.14 17. 120,096

PP 0.25-7.8 6.2 17. 2,700 MeO-Suc- Ala-Ala-Pro-Met-NA HL 0.1-2.5 2.4 0.72 300 MeO-Suc- Ala-Ala-Pro-Met(O)-NA’ HL 2.0 N.R.’ MeO-Suc- Ala-Ala-Pro-Met(O)-NA” HL 2.0 N.R. MeO-Suc- Ala-Ile-Pro-Met-NA HL 0.1-1.0 1.7 6.8 4,ooo

o Data of Zimmerman and Ashe (1977). b The sulfoxide diastereomer with RF of 0.50 upon thin layer chromatography on Silica Gel G using CHCls/methanol (5:1, v/v). ’ N.R., no reaction. ‘The diastereomer with RF of 0.65.

TABLE II Kinetic constants for the hydrolysis of synthetic I-nitroanilide substrates by human leukocyte cathepsin G

Conditions were: pH 7.50, 0.1 M Hepes buffer, 0.5 M NaCl, 9.8% dimethylsulfoxide at 25’C. Substrate Substrate cuncentra- -

tion range K”! k <a, L/Km P:, p4 PA Pn P,

m&l lTl.M s-’ M-I ,$-I

HBr . H-Gly-Leu-Phe-NA 0.2-2.2 21 0.43 20 Sue-Gly-Leu-Phe-NA 0.1-2.2 4.7 0.15 32

&u-Ala- Gly-Leu-Phe-NA 0.3-5.1 9.4 0.29 31 Sue- Ala- Ala- Phe-NA 0.3-2.7 3.9 1.5 380

Sue-Ala- Ala-Ala- Phe-NA 0.24-3.0 5.6 3.2 570 AC-Ala- Ala- Pro- Phe-NA 0.05-1.0 4.7 1.3 270

HCl. H-Ala- Ala- Pro- Phe-NA 0.07-1.4 4.1 0.47 110 MeO-Suc-Ala- Ala- Pro- Phe-NA 0.03-0.5 6.1 2.0 330

Sue-Ala- Ala-Pro- Phe-NA 0.2-3.5 2.9 3.1 1,100 Suc(4F)-Ala- Ala-Pro- Phe-NA 0.3-2.1 2.2 3.0 1,400

Glt-Ala- Ala-Pro- Phe-NA 0.2-3.1 2.6 1.7 660 Mal-Ala- Ala- Pro- Phe-NA 0.1-2.1 0.32 0.40 1,300

HBr. NHz(CH&CO-Ala- Ala-Pro- Phe-NA 0.1-2.2 13 3.2 250 Sue-Ala-Pro-Leu-Phe-NA” 1.3-8.5 7.2 0.064 8.9 Sue- Ala- Pro- Leu-NA 0.3-8.0 12 3.5 290

Sue-Ala- Ala-Pro- Leu-NA 0.1-3.3 3.4 1.3 380 MeO-Suc-Ala- Ala-Pro- Met-NA 0.1-2.1 0.31 0.52 1,700 MeO-Suc-Ala- Be- Pro- Met-NA 0.1-1.0 6.3 4.5 710 MeO-Sue-Ala- Ala-Pro- Met(O)-NA* 2.0 N.R.’

” The release of H-Phe-NA was measured using a ninhydrin assay. The reaction was carried out in a 0.04 M phosphate buffer, pH 7.50, containing 0.5 M NaCl and 9.6% dimethylsulfoxide at 37°C.

’ Either diastereomer. ’ N.R., no reaction.

TABLE III Kinetic constants for the hydrolysis of synthetic 4.nitroanilide substrates by bovine chymotrypsin

Conditions were: pH 7.50, 0.1 M Hepes buffer, 0.5 M NaC1, 9.8% dimethylsulfoxide at 25°C. -. Substrate

P5 PI P.1 P> PI

MeO- Suc- Ala- Ala- Pro-Val-NA MeO-Suc- Ala-Ala-Pro-Met-NA MeO-Sue-Ala-Ala-Pro-Met(O)-NA” MeO-Sue-Ala-Ala-Pro-Met(O)-NA* MeO-Suc- Ala- Ile- Pro-Met-NA

Sue- Ala- Ala- Pro-Phe-NA MeO-Sue-Ala-Ala-Pro-Phe-NA

- Substrate concentration range

lll.M 0.24-7.3 0.05-2.1

0.1-2.0 0.1-2.0

0.05-1.0 0.07-1.4 0.03-0.5

mM s-1

6.7 0.026 0.081 11 2.4 0.47 2.3 0.38 0.64 2.8 0.093 35 0.17 51

M-’ S-’

3.9 140,000

200 160

4,400 380,000 300,000

a The sulfoxide diastereomer with Rr of 0.50 upon thin layer chromatography on Silica Gel G using CHCL/methanol (5:1, v/v). b The diastereomer with RF of 0.65.

DISCUSSION tetrapeptides in which the P, residue was varied (Zimmerman and Ashe, 1977). The best 4-nitroanilide reported was Ac-Ala-

The extended substrate binding site of HL elastase has Ala-Pro-Val-NA and the most effective inhibitor was MeO- previously been investigated using peptide chloromethyl ke- Sue-Ala-Ala-Pro-ValCH&l. The latter was shown to be spe- tones (Powers et al., 1977) and 4-nitroanilide derivatives of citic for HL elastase and would not react with cathepsin G

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4030 Subsite Mapping of Cathepsin G and Leukocyte Elastase

even after incubation for several days. We have now shown that MeO-Sue-Ala-Ala-Pro-Val-NA is an excellent substrate for HL elastase. The change from an acetyl to a MeO-Suc- group results in a 2-fold improvement in both &at and Km. With both the chloromethyl ketone and the nitroanilide, the solubility was also increased by this change. Clearly HL elastase has a Ss subsite since the change from a Pb acetyl to a MeO-Suc- results in a significant rate acceleration in the inhibition by chloromethyl ketones and in the hydrolysis of 4- nitroanilides. The results also again demonstrate that the reactivity of a serine protease toward a substrate can be predicted on the basis of experiments with chloromethyl ke- tone inhibitors,or vice uersa (Powers, 1977).

Cathepsin G, the other major neutral protease of human leukocytes, has thus far proved to be fairly unreactive toward both peptide chloromethyl ketones or 4nitroanilide sub- strates. The most effective chloromethyl ketone inhibitor of cathepsin G (Z-Gly-Leu-PheCH&l) had a kow/[l] of 30 times less than the k,w/[l] of the best inhibitor with HL elastase (Powers et al., 1977). With tetrapeptide 4-nitroanilide sub- strates, the k,,JK,,, value for the best cathepsin G substrate was 175fold smaller when compared with HL elastase and 275fold smaller when compared with bovine chymotrypsin (Zimmerman and Ashe, 1977). The low rates of substrate hydrolysis or inhibition could be explained if the substrates or inhibitors investigated thus far with cathepsin G were not ideally matched to the enzyme’s extended substrate binding site in some manner. Thus, we decided to see if catalytic efficiency of cathepsin G could be improved by alterations in the substrate structure. Examination of data in Table II shows that we were in fact able to obtain a 6-fold improvement in k,,/K,,, when compared to the previous best substrate Ac-Ala- Ala-Pro-Phe-NA. However, HL elastase is still 7O-fold more reactive toward its best substrate than is cathepsin G toward its best substrate. This difference is primarily in kcat. The Km values with HL elastase and cathepsin G are very similar. In fact the K,,, values for HL elastase hydrolysis of MeO-Suc- Ala-Ala-Pro-Val-NA and the cathepsin G hydrolysis of MeO- Sue-Ala-Ala-Pro-Met-NA are within a factor of 2 of each other. However, the corresponding kcat values differ by 30- fold. Thus it appears that substrates bind equally well to the two enzymes; however, cathepsin G has a lower turnover rate. This could be due to a significant difference in the geometry of the catalytic residues of the two enzymes. Alternatively, there may be a requirement for some as yet undiscovered structural feature in a cathepsin G substrate which alters either the conformation of the enzyme or the structure of the E . S complex with a resulting increase in kc&t.

Cathepsin G clearly recognizes at least 5 residues (P5 to PI) of a substrate. In fact, changes in the nature of the PS group result in rather significant changes in k,.,/K,. The SE subsite of cathepsin G most likely contains a positively charged resi- due since substrates with negatively charged Ps groups (Sue-, Suc(4F)-, Glt-, Mal-) have higher k&K, values than those with no charges (AC- or MeO-Sue) or those with a positive charge. The Sz subsite prefers a Pro over Ala which is in turn preferred over Leu. A Pro residue cannot be accom- modated at Ps since Sue-Ala-Pro-Leu-Phe-NA was hydro- lyzed at the Leu-Phe bond instead of at the Phe-NA bond, even though cathepsin prefers substrates with S1 Phe over Leu. This P3 proline effect has been observed in a number of other serine proteases such as bovine chymotrypsin (Segal, 1972) and porcine pancreatic elastase (Thompson and Blout, 1973). It is caused by the interference of a PS Pro residue with the antiparallel /I sheet structure formed between a section of the extended substrate binding site of a serine protease and the peptide chain of its substrate (Segal et al., 1971).

The better substrates for cathepsin G and the HL elastase were tested for their specificity. MeO-Suc-Ala-Ala-Pro-Val- NA was not hydrolyzed by cathepsin G. Our sample of ca- thepsin G in fact hydrolyzed the substrate slowly but at a rate that could be caused by a 0.007% contamination by HL elastase. The hydrolysis was completely abolished when the cathepsin G sample was preincubated with the specific elas- tase inhibitor MeO-Sue-Ala-Ala-Pro-ValCH&l. Likewise, Sue-Ala-Ala-Pro-Phe-NA was hydrolyzed by HL elastase at a rate that could have been caused by a 3% contamination with cathepsin G. This hydrolysis was also completely abol- ished if the HL elastase sample was preincubated with the specific cathepsin G inhibitor Z-Gly-Leu-PheCH&l. Thus MeO-Sue-Ala-Ala-Pro-Val-NA and Sue-Ala-Ala-Pro-Phe-NA are completely specific for HL elastase and cathepsin G, respectively. These tools should be useful for the study of the biological role of these two enzymes.

As indicated earlier, the reactive site sequence of plasma al-protease inhibitor (aI-antitrypsin) has recently been shown to contain a P1 methionine residue (Johnson and Travis, 1978). Based on this report we synthesiztd two tetrapeptide 4-nitroanilides MeO-Sue-Ala-Ala-Pro-Met-NA and MeO-Suc- Ala-Ile-Pro-Met-NA. The latter has the exact sequence of the Pq to PI residues at the al-PI inhibitor reactive site. Both of the compounds bind to and are hydrolyzed by HL elastase, cathepsin G, and bovine chymotrypsin, three enzymes which are known to be inhibited by al-PI. With cathepsin G, in fact, one of the tetrapeptides with a Met at P1 is the best cathepsin G 4-nitroanihde substrate thus far discovered. With HL elas- tase the Met tetrapeptides do not have as high k,,JK, values as those with P1 Val residues, but are still reasonably reactive. Trypsin (bovine), another enzyme inhibited by al-PI, did not, however, hydrolyze either Met tetrapeptide.

The mechanism of inhibition of serine proteases by al-PI has not yet been worked out in detail. Undoubtedly the enzyme first binds to al-PI due to recognition of a certain sequence of amino acids with a favorable geometry at the reactive site of al-PI. X-ray studies with trypsin-trypsin inhib- itor (soybean and pancreatic) complexes have shown that the majority of interactions occur between the Pq to P3’ residues of the inhibitor and the enzyme’s active site (Sweet et al., 1974; Huber et al., 1974). By analogy it would be expected that the important interactions between al-PI and various series proteases involve the P4 to Ps’ residues of the al-PI reactive site. Our studies with MeO-Sue-Ala-Ile-Pro-Met-NA show that the Pq to PI sequence of al-PI is sufficient to get recognition by, and reaction with, HL elastase and cathepsin G, the two major proteases of the granules of leukocytes. Obviously, interactions with Pr’ to Ps’ residues would increase the strength of the interaction. In addition, the reactive site of al-PI is probably locked in a conformation which complements the structure of the extended substrate binding-region of serine proteases, while the tetrapeptides have considerable conformational flexibility in solution which would decrease the magnitude of the binding interaction. The conformation of the al-PI must be important for reaction with trypsin since the sequence (at least the Pq to Pi sequence alone) seems insufficient to obtain recognition.

Most people with emphysema have a past history of ciga- rette smoking. It has been postulated that smoking in some way affects the balance between free leukocyte proteases and their inhibitors with the result being proteolysis of the lung due to the presence of excess proteases. Since al-PI is consid- ered to be the major inhibitor of leukocyte proteases, it is reasonable to postulate a link between al-PI inactivation and lung proteolysis. In fact, Janoff and Carp (1977) have shown that cigarette smoke condensation will inactivate al-PI and

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Subsite Mapping of Cathepsin G and Leukocyte Elastase 4031

postulate the presence of damaging oxidizing agent(s) since Hance, A. J., and Crystal, R. G. (1975) Am. Reu. Resp. Dis. 112,656- the inactivation was prevented by antioxidants. In the preced- 711 ing paper Johnson and Travis (1979) show that oxidation of Huber, R., KukIa, D., Steigemann, W., Deisenhofer, J. and Jones, A. al-PI at Met leads to inactivation of the inhibitor. We have (1974) in Buyer Symposium V: Proteinase Inhibitors (Fritz, H.,

shown that neither diastereomeric sulfoxide of MeO-Suc-Ala- Tschesche, H., and Greene, L. J. eds) pp. 497-512, Springer-Verlag, New York

Ala-Pro-Met(O)-NA is hydrolyzed by HL elastase, cathepsin G or bovine trypsin, whereas MeO-Sue-Ala-Ala-Pro-Met-NA

Janoff, A., and Carp, H. (1977) Am. J. Resp. Dis. 116.65-72 Johnson, D. A., and Travis, J. (1976) Biochem. Biophys. Res. Com-

is an excellent substrate for both HL elastase and cathepsin mun. 72,33-39

G. Neither sulfoxide acts as a competitive inhibitor of the Johnson, D. A., and Travis, J. (1978) J. Biol. Chem. 253.7142-7144

elastase hydrolysis of MeO-Sue-Ala-Ala-Pro-Val-NA, nor the Johnson, D., and Travis, J. (1979) J. Biol. Chem. 254,4022-4026

cathepsin G hydrolysis of Sue-Ala-Ala-Pro-Phe-NA, indicat- Lestienne, P., Dimicoh, J.-L., and Bieth, J. (1977) J. Biol. Chem. 262,

ing that the oxidation from sulfide to sulfoxide is enough to 5931-5933

Lestienne, P., Dimicoli, J.-L., and Bieth, J. (1978) J. Biol. Chem. 253, prevent binding of these tetrapeptides to both HL elastase 3459-3460 and cathepsin G. Bovine chymotrypsin will still react with the Mittman, C., ed (1972) Pulmonary Emphysema and Proteolysis, pp. sulfoxides, but both K,,, and kcat are poorer than with the l-537, Academic Press, New York methionine derivative. Thus one can conclude that oxidation Powers, J. C. (1977) in Chemistry and Biochemistry of Amino Acids,

of the Pi Met residue of al-PI’s reactive site to a sulfoxide is PeDtides and Proteins. (Weinstein. B.. ed) DD. 65-178. Marcel . . .

sufficient to destroy this inhibitor’s capacity for inhibiting Dekker, New York

most serine proteases. Oxidation of &,-PI by some component Powers, J. C., and Gupton, B. F. (1977) Methods Enzymol. 46, 208-

216 in cigarette smoke would offer one explanation in molecular Powers, J. C., Gupton, B. F., Harley, A. D., Nishino, N., and Whitley, terms for the link between smoking and emphysema. Whether R. J. (1977) Biochim. Biophys. Acta 485, 156-166 this reaction, in fact, takes place in uiuo remains to be inves- Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun.

tigated. 27, 157-162

Segal, D. M. (1972) Biochemistry 11, 349-356 SegaI, D. M., Powers, J. C., Cohen, G. H., Davis, D. R., and Wilcox,

Acknowledgments-We thank Brian McRae for measuring the P. E. (1971) Biochemistry 10,3728-3738

kinetics of hydrolysis of Sue-Ala-Pro-Leu-Phe-NA by cathepsin G. Starkey, P. M. (1977) in Proteinases in Mammalian Cells and

The HL elastase and cathepsin G used in this study were generous Tissues (Barrett, A. J., ed) pp. 57-89, Elsevier/North-Holland

gifts of Dr. James Travis and his group at the University of Georgia. Biomedical Press, Amsterdam

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K Nakajima, J C Powers, B M Ashe and M Zimmerman1-protease inhibitor reactive site.

leukocyte elastase. Studies with peptide substrates related to the alpha Mapping the extended substrate binding site of cathepsin G and human

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