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

Abstract

Using molecular dynamics simulations, we examined the plausible conformations for a hexaNAG substrate 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) involves substrate distortion, (ii) the first step of acid-catalyzed hydrolysis (protonation of the linking anomeric oxygen between GlcNAc residues −1 and +1) requires a boat conformation for the GlcNAc residue at binding subsite −1; (iii) ab initio quantum mechanical calculations (HF/6-31G**) predict that protonation of a GlcNAc in a boat conformation leads to spontaneous anomeric bond cleavage to yield an oxazoline ion intermediate. We also studied several conformations of two possible hydrolysis intermediates: the oxocarbenium ion and the oxazoline ion. Only the oxazoline ion orients in the enzyme active site so as to allow stereoselective attack by water. This leads to retention of configuration in the anomeric product as observed experimentally. It is pos...