Structural Change in -Chymotrypsin Induced by Complexation with
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- 作者：Elena S. Stavridi ; Kevin O'Malley ; Christine M. Lukacs ; William T. Moore ; John D. Lambris ; David W. Christianson ; Harvey Rubin ; and Barry S. Cooperman
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：August 20, 1996
- 年：1996
- 卷：35
- 期：33
- 页码：10608 - 10615
- 全文大小：469K
- 年卷期：v.35,no.33(August 20, 1996)
- ISSN：1520-4995
文摘
Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr)proteolyze Chtr withinthe complex that Chtr forms with antichymotrypsin (ACT). As freeChtr is stable both to self-digestionand to digestion by HNE, these results are indicative of a stabilityand/or conformational change in Chtrthat accompanies complex formation. As determined by bothN-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), themajor initial sites of HNE cleavageof complexed Chtr are between 
-chain residues A158/S159 andV188/S189. Significantly, this lattersite is at the base of the S1 site that recognizes the P1 position ofthe serpin. A slower cleavage in the
-chain between T139/G140 is also found. In addition, rACT iscleaved between residues V22/D23.The -chain of complexed Chtr is also cleaved by free Chtr, butat different sites: L162/L163 and W172/G173. -Chain cleavages were also found between residues Q81/K82and F114/S115. Cleavages similarto those described above were also found when Chtr was complexed withthe L358F-rACT variant, butnot for Chtr complexed with either of the smaller inhibitors bovinepancreatic trypsin inhibitor or turkeyovomucoid third domain, nor for the covalent adduct of Chtr withN-p-tosylphenylalanyl chloromethylketone. We conclude that the structural change in Chtr making it aproteinase substrate is coupled withthe large conformational change in ACT following complex formation.Complexed Chtr is much lessreactive toward proteolytic digestion in the presence of high salt thanin its absence, in accord with thehigh-salt induced release of active enzyme from the Chtr·rACTcomplex and the suggestion that electrostaticinteractions mediate the coupling of structural change between rACT andChtr within the Chtr·rACTcomplex. Potential physiological consequences of this work areexplored.
-chain residues A158/S159 andV188/S189. Significantly, this lattersite is at the base of the S1 site that recognizes the P1 position ofthe serpin. A slower cleavage in the-chain between T139/G140 is also found. In addition, rACT iscleaved between residues V22/D23.The -chain of complexed Chtr is also cleaved by free Chtr, butat different sites: L162/L163 and W172/G173. -Chain cleavages were also found between residues Q81/K82and F114/S115. Cleavages similarto those described above were also found when Chtr was complexed withthe L358F-rACT variant, butnot for Chtr complexed with either of the smaller inhibitors bovinepancreatic trypsin inhibitor or turkeyovomucoid third domain, nor for the covalent adduct of Chtr withN-p-tosylphenylalanyl chloromethylketone. We conclude that the structural change in Chtr making it aproteinase substrate is coupled withthe large conformational change in ACT following complex formation.Complexed Chtr is much lessreactive toward proteolytic digestion in the presence of high salt thanin its absence, in accord with thehigh-salt induced release of active enzyme from the Chtr·rACTcomplex and the suggestion that electrostaticinteractions mediate the coupling of structural change between rACT andChtr within the Chtr·rACTcomplex. Potential physiological consequences of this work areexplored.