Substrate Distortion to a Boat Conformation at Subsite -1 Is Critical in the Mechanism of Family 18 Chitinases

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• 作者：Ken A. Brameld and William A. Goddard ; III
• 刊名：Journal of the American Chemical Society
• 出版年：1998
• 出版时间：April 22, 1998
• 年：1998
• 卷：120
• 期：15
• 页码：3571 - 3580
• 全文大小：207K
• 年卷期：v.120,no.15(April 22, 1998)
• ISSN：1520-5126

文摘

Using molecular dynamics simulations, we examined the plausibleconformations for a hexaNAGsubstrate bound to the active site of Chitinase A. We find that(i) the hydrolysis mechanism of Chitinase A(a family 18 chitinase from Serratia marcescens) involvessubstrate distortion, (ii) the first step of acid-catalyzed hydrolysis (protonation of the linking anomeric oxygen betweenGlcNAc residues -1 and +1) requiresa boat conformation for the GlcNAc residue at bindingsubsite -1; (iii) ab initio quantum mechanicalcalculations(HF/6-31G**) predict that protonation of a GlcNAc in a boatconformation leads to spontaneous anomericbond cleavage to yield an oxazoline ion intermediate. We alsostudied several conformations of two possiblehydrolysis intermediates: the oxocarbenium ion and the oxazoline ion.Only the oxazoline ion orients in theenzyme active site so as to allow stereoselective attack by water.This leads to retention of configuration inthe anomeric product as observed experimentally. It is possiblethat all family 18 chitinases share a commonmechanism. Hence, we suspect that distortion of the substrate intoa boat form at subsite -1 is required forany glycosyl hydrolase which has only one acidic residue in the activesite. The design of an inhibitor forthese systems based on the boat distorted sugar conformation isdiscussed.