Substrate binding modes and anomer selectivity of chitinase A from Vibrio harveyi.

High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites -2 to +2 over subsites -3 to +2 and pentaNAG only required subsites -2 to +2, while subsites -4 to +2 were not used at all by both substrates. The results suggested that binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode (-2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline alpha chitin was initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward beta substrates. The Trp275 mutation modulated the kinetic property of the enzyme by decreasing the catalytic constant (k (cat)) and the substrate specificity (k (cat)/K (m)) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2), thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive degradation toward insoluble chitin.