Kinetic Studies on the Hydrolysis of N-Acetylated and N-Deacetylated Derivatives of 4-Methylumbelliferyl Chitobioside by the Family 18 Chitinases ChiA and ChiB from Serratia marcescens

Abstract

Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)₂-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)₂-UMB (4)] by ChiA and ChiB from Serratiamarcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K_m value relative to 1, indicating that the N-acetyl group was recognised at the –2 subsite. The k_cat value obtained with ChiA was identical to the k_cat value observed for 1. However, the k_cat value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K_m values relative to 1, respectively, and 60- and 30-fold smaller k_cat values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.