Importance of Tetrahedral Intermediate Formation in the Catalytic Mechanism of the Serine Proteases Chymotrypsin and Subtilisin

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• 作者：Teodolinda Petrillo ; Catrina A. O鈥橠onohoe ; Nicole Howe ; J. Paul G. Malthouse
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：August 7, 2012
• 年：2012
• 卷：51
• 期：31
• 页码：6164-6170
• 全文大小：295K
• 年卷期：v.51,no.31(August 7, 2012)
• ISSN：1520-4995

文摘

Two new inhibitors in which the terminal 伪-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for 伪-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3鈥?-fold while a glyoxal group increases the level of binding by 500鈥?000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000鈥?29000 and 101000鈥?59000 for 伪-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK_a of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.