Human neutrophil proteinase 3: Mapping of the substrate binding site using peptidyl thiobenzyl esters

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<ul><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS November 16, 1992 Pages 1318-1324 </p><p>EcJMANNgIITBopHILpBoTgeJBsE3: MAPPmGOFTBg SUBSTRAl!E BINDING Sl!lR U!SING PRPTIDYLTHIoBKNzyLRs!rRRs </p><p>Michael J. Brubakerl, William C. Groutas*#, John R Hoidal@, and Narayanam v. Rae@ </p><p>//Department of Chemistry, Wichita State University, Wichita, Ransas 67208 </p><p>@Division of Respiratory, Critical Care and Occupational h&amp;dicine, </p><p>University of Utah Health Sciences Center, Salt Lake City, Utah 84132 </p><p>Received August 14, 1992 </p><p>&amp;lmmzuy A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steadydte kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active titez (a) the preferred PI residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valise or alanine (in decreasing order of preference), 01) the enzyme has an extended active site, and (c) its active site is similar to that of the related serine proteinaws leukocyte elaskse and leukocyte cathepsin G. 0 1992 Academic Press, Inc. </p><p>Proteinase 3 (PR-3) (JLC. 34.21.-) is a serine endopeptidase that has </p><p>been isolated recently from the granulea of polymorphonuclear leukocytes </p><p>(1). Prehminaq studies related to its characterization (2), inhibition (3) </p><p>and cloning (4) have been described. An increasing body of evidence </p><p>indicates that PR-3 may play a major role in the pathophysiology and/or </p><p>etiology of some neutrophil-mediated diseases, including Wegener% gran- </p><p>ulomatosis (56), pulmonary emphysema (l), psoriasis (7), and cystic </p><p>fibrosis (8). Rqually important is the recent observation that PR-3 is </p><p>identical in myeloblastin, an enzyme involved in the growth and differ- </p><p>entiation of human leukemic cells (9). In order to probe the active site </p><p>*To whom correspondence should be addressed. </p><p>0006-291X/92 $4.00 </p><p>Copyright 0 1992 by Academic Press, Inc. All rights qf reproduction in an): form reserved. 1318 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>of PR-3 and to place the future design of inhibitors on a more secure </p><p>biochemical footing, we have utilized a series of peptidyl thiobonzyl </p><p>esters to map the active site of PR-3 and the results of our studies are </p><p>described herein </p><p>Reagents The peptides listed in Table I were synthesized using s&amp;ndard peptide methodology. The purity of all compounds was checked by thin- Layer chromatography on silica gel plates. The NMR spectra were consistent with the assigned structures and were determined by using a Varian XL300 Fourier transform spectrometer. Rlemental analyses were performed by MXIW Laboratories, Phoenix, AZ Gptical rotations were recorded using a Jasco DIP360 polerimeter. A Gilford uv/vis spectr+ photometer was used in the enzyme kinetics studies Amino acid derivatives and dipeptides used in syntheses were purchased from Rachem Galifornia Co. or Sigma Chemical Ca, St Louis </p><p>Er\zwe Proteinase 3 was isolated as described previously (1). In a representative kinetics assay, 50 pl of a 16 niM aalntion of 5,5- ditbi~bj&amp;%nitrobenzoic acid) (DTNR) in dimethyl sulfoxide were mixed in a thermostated cuvette (25C) with 50 pl MeG-Stu-Nva-SBzl(649 m.M) in dimethyl sulfoxide and 600 ~1 of HRPR!3 buffer (61 M, 65 Bd NaGl, pH 7.2). A 100 ul aliquot of protew 3 (0.357 JIM) was then added and the change in abeorbance was monitored at 412 nm for one minuti The concentration of DIKSG (10%) was kept constant for all tdmtratea </p><p>-Yd </p><p>v The kinetic constants, Km and k-t, were obtained by determining the initial velocity in duplicate at four to six sub&amp;rate concentrations. Initial velocities were calculated by a linear least-squares fit of the experimental data. For each substrate, kat and K, were calculated by nonlinear least-squares fit of the initial velocity data to the MiehaelisMenten equation using the program RNKPPLTKR (Riosoft, Cambridge, UK, version 1.03). Doubl~reciprocal plots were linear in all cases </p><p>The kinetic constants for the hydrolysis of a series of peptidyl </p><p>thiobenzyl ester substrates by proteinasc+ 3 are given in Table L For </p><p>comparative pmpoms, and when available, the corresponding k&amp;Km </p><p>values with human leukocyte elastase (HLE) are also listed in Table L </p><p>1319 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>TABLE 1. Steady-State Kinetic Parameters for the Proteinase 3-catalyzed Hydrolysis of </p><p>Peptidyl Thiobenzyl Estersa </p><p>Compound k cat Kn Lt/Kn s-1 ml4 M-4 s-' </p><p>p, p, p, P,b </p><p>1 BCC-Ala-pNA </p><p>2 BCC-Ala-ONp 8 </p><p>3 BCC-Ala-SBzl 2 </p><p>4 HeOSuc-Ala-SBzl 5 </p><p>5 MeOSuc-Abu-SBzl 17 </p><p>6 MeOSuc-Val-SBzl 5 (10) </p><p>7 MeOSuc-Nva-SBzl 19 </p><p>8 MeOSuc-Pro-Ala-SBzl 9 </p><p>9 MeOSuc-Pro-Abu-SBzl 15 </p><p>10 HeOSuc-Ala-Pro-Ala-SBzl 10 (46) </p><p>11 MeCSuc-Ala-Pro-Abu-SBzl 9 </p><p>12 MeCSuc-Ala-Ala-Pro-Ala-SBzl 11 (53) </p><p>13 MeDSuc-Ala-Ala-Pro-Abu-SBzl 11 </p><p>C </p><p>0.47 17,000 </p><p>0.48 4,200 </p><p>0.50 10,000 </p><p>0.25 68,000 </p><p>0.12 41,700 (0.015) (680,OOO)d </p><p>0.30 63,300 </p><p>0.89 10,100 </p><p>0.25 60,000 </p><p>0.12 83,300 (0.012) (3,840,000)d </p><p>0.09 100,000 </p><p>0.05 220,000 (0.13)(4,100,000)d </p><p>0.04 275,000 </p><p>a Conditions used: 0.1 I HEPZS buffer, 0.5 H NaCl, pH 7.2, 10% DHSO, 25' C; [DTNB]: 0.80 a. "Nomenclature: S,, S,, S,, . ..S. and S1#, S,', S,',..S.' correspond to the enzyme subsites on either side of the scissile bond. Each subsite accommodates an amino acid side chain designated P,, P,, P,,...P. and P,#, P,', P,',..P,' of the substrate or inhibitor. Sx is the primary specificity site (Schecter 8 Berger, 1967). CNo reaction. ' k,,,/K" values for human leukocyte elastase taken from Stein et al., 1987. </p><p>The data cited in Table I are supportive of the following infer- </p><p>ences regarding the substrate specificity of PR-3: </p><p>(a) The preferred P, residue of the S, sub&amp;e of PR3 is a mnnll_ </p><p>hydrophobic amino acid such as Abu, NVa, Val, and Ala. The best mom+ </p><p>meric substrates had a two or thr e-carbon straight alkyl sideshain </p><p>(compounds 5 and 7). kcat increased three to four-fold and K, decnwed </p><p>1320 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>two-fold in going from a P, residue with a one-carbon 4, two-carbon 5, </p><p>and threeear bon 7 side chain Ibis is in accord with the resulb of </p><p>recent studies using monomeric inhibitors, where inhibitory potency </p><p>(expressed as the biomolecular rate constant k,,JI hf s-l) correlated </p><p>closely with the length of the P, side chain (3). It is interesting to note </p><p>that the correspondiug kcat and Km values for compound 6 with ELR </p><p>are 2-f old higher and g-fold lower, respectively, reflecting the greater </p><p>catalytic efficiency of HLR over PR-3 in hydrolyzing thiobenzyl ester </p><p>substratea </p><p>(b) Like HLR, the introduction of proline at the P, position had </p><p>no significant effect on the magnitude of the kinetic parameters </p><p>(compounds 5 and 9). It is likely that the presence of proline simply </p><p>permits a peptidyl substrate or inhibitor to assume a geometry that is </p><p>complementary to the hemispherical shape of the active site (Figure 1). </p><p>(c) &amp;tension of the peptidyl chain to three or four residues </p><p>affords a modest but steady improvement in the strength of binding, </p><p>without any improvement in kat (compare, for example, compounds 9, </p><p>11, and 13). With the exception of the monomeric substrates, the </p><p>magnitude of kat remains constant at about 11 s-l. This is nearly </p><p>identical to the corresponding kcat values obtained with HLR (11). </p><p>Significantly, the corresponding Km values with HLR are five- to ten- </p><p>fold lower. Peptide chain length, allowing the operation of remote </p><p>subsite interactions in the serine proteinases, has been shown to augment </p><p>catalytic efficiency, activate the operation of the chargerelay system </p><p>and to regulate P, specificity (11-13). This is evidently true in the case </p><p>of PR-3 as well, although how P, specificity is regulated by peptide </p><p>length in PR-3 has not been addressed in this study. </p><p>(d) PR-3 is highly homologous to ela&amp;ase (54%) and cathepsin G </p><p>(35%) (4). We have used the kn own x-ray crystal structure of HLR (14) </p><p>to construct the active site of the enzyme (shown in Figure 1X The </p><p>horseshoe shape of the active site and the catalytic residues are clearly </p><p>evident. We then used the known amino acid sequence of PR3 to bigh- </p><p>1321 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>0 PHE-192 El n PHE-192 A LYS-192 \ </p><p>SER 195 </p><p>ASP102 </p><p>* </p><p>Fipnre Amino acid reaiduea at the active site of human 1-e . elastaae and the putative active site of proteinas&amp;. The co- amino acid rtxiduea for human leukocyte cathepain G have alao been included for comparative pnrposee. </p><p>light the Gmilarity between the putative active site of PR-3 and that </p><p>of HLE. The observed simikity in the substrate specificities of PR-3 </p><p>and ISLE reflects a similar makeup of their active sites. A noteworthy </p><p>difference between HLE and PR3 ia in residue 213 (Figure 1). Clearly, </p><p>other subtle differences in the geometry of the active sitea of PR3 and </p><p>HLE may account for their difference8 in catalytic prowess. </p><p>1322 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>(e) The greater sensitivity, stability, and volubility of the readily- </p><p>accessible monomeric thiobenzyl ester substrates g-7 make their uee in </p><p>assaying Prote inase-3 activib distinctly advantageous over the </p><p>hydrolytically-unstable pnitrophenol e&amp;era Furthermore, incorporation </p><p>of the methoxysuccinyl group yields substrates with greater aqueous </p><p>solubility. </p><p>In conchion, the active site of PR-3 baa been mapped using a </p><p>series of peptidyl thiobenzyl ester substratea PR-3 has an extended, </p><p>horseshotAmped binding site and prefers a P, residue with a two- or </p><p>threecarbon side chain. Given the relatively high abundance of PR-3 </p><p>in neutrophils, its ability to degrade a variety of matrix components, and </p><p>the fact that PR-3 is not inhibited by secretory leukoprotease inhibitor </p><p>(l), the results of the present study describing characteri&amp;.ica of the </p><p>active site should provide a rational basis upon which to begin to </p><p>develop effective inhibitors for studies of the physiological and </p><p>pathophysiological roles of the enzyme </p><p>The generous financial support of the National Institutea of Health @IL 38048) is gratefully acknowledged. </p><p>1. </p><p>2. </p><p>3. </p><p>4. </p><p>5. 6. </p><p>7. </p><p>Kao, RC., Wehner, N-G., Skubitz, KA&amp; Gray, m k Hoidal, J.R. (1988) m 8s: 196%1973. Rao, N-V., Webner, N.G., Marshall, B.C, Gray, W.R., Gray, B.H. % Hoidal, JX (1991) d. m 954@9548, Groutas, W.C., Hoidal, JX., Brubaker, I&amp;J., Stanga, MA., Venkataraman, R., Gray, B.H. &amp; Rae, N.V. (1990) J. 3&amp; 108!5-1087 Campanelli, D, Melchior, M, Fu, Y, Nakata, hi., Shaman, &amp; Nathan, C. &amp; Gabay, JX (1990) J. lTz, 1709171!5. Jennette, J.C, Hoida&amp; J-R L Falk, R.J. (1990) Blppd 75, -2264. Jenne, D.E., Tschopp, J, Ludeman, J, Utecht, B. &amp; Gross, WI.,. w9Q Natnre 346 520. Wiedow, 0, Ludemanq J. &amp; Utecht, B. (1991) w Res 174 6-10. </p><p>1323 </p></li><li><p>Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS </p><p>8 9. </p><p>10. </p><p>11. </p><p>12 13. 14. </p><p>15. </p><p>G&amp;t&amp;, W. &amp; Doring, G. (1986) Am. 134, 4956. . Labbaye, C, Musette, P. &amp; Cayre, YJ% (1991) m </p><p>UsA ss, 9253-9256. Digeuis, GA., Agha, B.J, Tsuji, K., Kato, 116 &amp; Shinogi, A#. (1966) J.Med 29, 14681476. Stein, RL, Strimpler, AM., Hori, E k Powers, J.C. (1967) </p><p>. . mx&amp;mf@z zs, 1301-1305. stein, EL. (198!5) J. 107, 5767-5775. Stein, ILL. (1985) Arch. 236, 677-m Navia, WA., McKeever, BM., Springer, J9, Lin, T-Y, Williamq IX&amp; Fluder, EM, Darn, C.P. &amp; Haogsteen, K (1969) &amp;x,-&amp;&amp;L k&amp;ad sci. u&amp;j ss, 7-11. Scbecter, I. &amp; Berger, A (1967) Q 16l, 143-14s. </p><p>1324 </p></li></ul>

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