Reaction Mechanism of Chitinase Belonging to Glycosidase Family 18

Abstract

To determine the reaction mechanism of ChiA from Serratia marcescens, we synthesized the partially and fully N-deacetylated 4-methylumberlliferyl chitobioside (1) derivatives, GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)₂-UMB (4) as fluorogenic ChiA substrates and performed kinetic analyses of their hydrolysis by ChiA. With the exception of compound 4, ChiA released UMB from all compounds tested. The S-v curves of ChiA hydrolysis of 1 exhibited atypical kinetic patterns, which may have been due to the formation of enzyme complexes with multiple molecules of the substrate. Conversely, the S-v curves generated during ChiA cleavage of 2 and 3 exhibited typical Michaelis-Menten profiles. Compared with enzyme hydrolysis of 1, hydrolysis of 2 resulted in a 14-fold higher K_m value, indicating that the N-acetyl group was recognized at the -2 subsite. The k_cat value obtained with ChiA was identical to the k_cat value observed for 1. Compared with the ChiA hydrolysis of 1, ChiA hydrolyzed 3 with a 5-fold greater K_m and a 60-fold smaller k_cat. The reaction mechanism of family 18 chitinases is discussed based on the results obtained from the hydrolysis of these compounds.