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    The importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin
    Two new inhibitors have synthesized where the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced by a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H respectively. Using these inhibitors we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases inhibitor binding 3-4 fold while a glyoxal group increases binding by 500-2000 fold. We show that at pH 7.2 the effective molarity of the catalytic hydroxyl group of the active site serine is 41,000-229,000 and 101,000 to159,000 for α-chymotrypsin and subtilisin Carlsberg respectively. It is estimated that oxyanion stabilisation 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 pKa of the active site histidine enabling 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.
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