Multiple Roles of Asp313 in the Refined Catalytic Cycle of Chitin Degradation byVibrio harveyiChitinase A

Three acidic residues in the DXDXE sequence motif are suggested to play a concerted role in the catalysis of Vibrio harveyi ChiA. An increase in the optimum pH of 0.8 units in mutant D313A/N indicates that Asp313 influences the pKa of the ionizing groups around the cleavage site. D313A showed greatly reduced kcat/Km and increased KD, suggesting that Asp313 participates in catalysis and ligand binding. Investigation of the enzyme-substrate interactions of V. harveyi ChiA and Serratia marcescens ChiB revealed two conformations of Asp313 and (-1)GlcNAc. The first conformation, likely to be the initial conformation, showed that the β-COOH of Asp313 only interacted with the -C=O of the N-acetyl group in the distorted sugar. The second conformation, formed from the first by concerted bond rotations, demonstrated hydrogen bonds between the Asp313 side chain and the -NH of the N-acetyl group and the γ-COOH of Glu315. Here we propose a further refinement of the catalytic cycle of chitin hydrolysis by family-18 chitinases that involves four steps: Step 1: Pre-priming. An acidic pair is formed between Asp311 and Asp313. Step 2: Substrate binding. The Asp313 side chain detaches from Asp311 and rotates to form a H-bond with the C=O of the 2-acetamido group of -1GlcNAc. Step 3: Bond cleavage. The side chain of Asp313 and the 2-acetamido group simultaneously rotate, permitting Asp313 to interact with the side chain of Glu315 and facilitating bond cleavage. Step 4: Formation of reaction intermediate. The transient (-1) C1-GlcNAc cation readily reacts with the 2-acetamido group, forming an oxazolinium ion intermediate. Further attack by a neighboring water results in retention of β-configuration of the degradation products.