Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid

Abstract

We have studied the kinetic mechanism for the production of product N-acetylglucosamine from the enzymatic hydrolysis of chitin oligosaccharides by the binary enzyme system, chitinase plus β-N- acetyl- d-glucosaminidase , isolated from pharate pupal moulting fluid of Manduca sexta (L.). A synergism occurs with the rate of monomer production by the enzyme mixture greater than the sum of the rates of the individual enzymes. For the production of monosaccharide from tetrasaccharide, pentasaccharide and hexasaccharide the time courses exhibited by a mixture of purified insect enzymes are essentially identical to those developed using crude moulting fluid. Satisfactory agreement with the experimental data is obtained when using theoretical calculations based on the following: (1) a random attack mechanism with no interaction between the two enzymes, (2) monosaccharide is not produced to a significant extent from the original substrate, (3) chitinase forms both productive and nonproductive complexes with substrate, (4) nonproductive substrate binding to chitinase is more favorable than productive binding, (5) chitinase forms multiple substrate nonproductive complexes, and (6) intermediate metabolite concentrations are low during the course of hydrolysis. β- N-Acetylglucosaminidase is dependent to a great extent on chitinase for production of small fragments from which monosaccharide can be released.