title: Multiple roles of Asp313 in the refined catalytic cycle of chitin degradation by Vibrio harveyi chitinase A.
abstract: 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.
authors.affiliation: Biochemistry-Electrochemistry Research Unit, School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand. wipa@sut.ac.th
authors.forename: Wipa
authors.initials: W
authors.lastname: Suginta
authors.forename: Natchanok
authors.initials: N
authors.lastname: Sritho
mesh_terms: D019943:Amino Acid Substitution; D001224:Aspartic Acid; D055162:Biocatalysis; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D012706:Serratia marcescens / Q000201:enzymology; D014733:Vibrio / Q000201:enzymology*
chemical_list: D002686:Chitin; D001224:Aspartic Acid; D002688:Chitinases