Thirty Years with Glucoamylase

Abstract

Five topics were picked up from our studies with Rhizopus glucoamylase during 30 years, and briefly introduced. 1) From the study of pH profile, it was concluded that the same pair of carboxylate and carboxyl groups, whose pKa's are 2.9 and 5.9 respectively, are involved in the catalytic hydrolysis of both a-1, 4 and a-1, 6 glucosidic linkages. 2) Calorimetric measurement of enthalpies of hydrolysis of a-1, 4 and a-1, 6 linkages revealed that heats of hydrolysis of a-1, 4 and a-1, 6 linkages are -1.1 kcal/mol and + 1.3 kcal/mol, respectively. Therefore, the reverse reaction, the condensation of the two linkages, is more favored, at least enthalpically, for a-1, 6 linkage formation, which is actually the case. 3) The number-average molecular weight of amyloses can be determined from the analysis of the time course of glucoamylase-catalyzed hydrolysis of amyloses. 4) Glucoamylase is the first enzyme to which the subsite theory was successfully applied. The subsite affinities of each subsite and the intrinsic rate constant of glucosidic linkage splitting have been determined solely from the rate parameters (Michaelis constants and molar activities) of a series of linear substrates. 5) Stopped-flow kinetic studies down to millisecond region on the binding of the enzyme with various substrates (maltooligosaccharides) and inhibitors (gluconolactone and 1-deoxynojirimycin) were done successfully. The results are useful in considering the detailed mechanisms of the enzyme reaction.