Rate Equation for Amylase-Catalyzed Hydrolysis, Transglycosylation and Condensation of Linear Oligosaccharides and Amylose

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Abstract
In order to analyze experimental results of amylase-eatalyzed reactions, which include transglycosylation and condensation as well as hydrolysis, with amylose and linear oligosaccharides as substrates, a general equation governing the concentrations of substrate and product species in a reaction mixture is derived based on the subsite theory. The equation is devised so as to follow the location in products of radioactive glucose which was originally located at a certain position in a substrate. The reaction scheme assumed is as follows: 1) Enzyme can bind any substrate reversibly to form an enzyme-substrate complex, ES. 2) From the ES complex, a glucosyl bond or C1-OH bond at the reducing end of the substrate is cleaved to from a reactive intermediate, releasing ja product of shorter chain or H2O. 3) The reactive intermediate binds an acceptor molecule reversibly to form a ternary complex from which the product of tranhsglycosylation or condensation is produced. The binding constants between enzyme and sybstrate, and between reactive intermediate anhd acceptor can be estimated from the subsite affinities. The rate constants of catalytic processes such as cleavage of a glucosyl bond and C1-OH bond, bydrolysis and transglycosylation are assumed to be independent of the degree of polymerization as well as of the binding mode of the substrate. The applicability of this scheme and rate equation was tested successfully with the Taka-amylase A-catalyzed reaction of maltohexaose labeled with 14C at the reducing end.