CONFIGURATIONAL SPECIFICITY - UNAPPRECIATED KEY TO UNDERSTANDING ENZYMIC REVERSIONS AND DE NOVO GLYCOSIDIC BOND SYNTHESIS .I. REVERSAL OF HYDROLYSIS BY ALPHA-, BETA- AND GLUCOAMYLASES WITH DONORS OF CORRECT ANOMERIC FORM

Theoretical considerations have led to a new approach for investigating enzymic glycosidic bond formation from free sugars. By use of substrates of defined anomeric form under conditions limiting mutarotation, evidence has been obtained that such condensations require donor substrates of specific configuration. Thus, crystalline glucoamylase from Rhizopus niveus has been found to catalyze the rapid synthesis of maltose and a slower synthesis of isomaltose specifically from β-d-glucopyranose. For the first time, the condensing and hydrolytic activities of this enzyme show a clear correspondence not revealed by past studies of glucosaccharide (mainly isomaltose) formation by prolonged glucoamylase action on mixtures of d-glucose anomers. Crystalline sweet potato β-amylase, likewise, has been found to catalyze the rapid synthesis of maltotetraose specifically from β-maltose, and crystalline hog pancreatic α-amylase the rapid synthesis of maltotetraose specifically from α-maltose. These condensations also confirm for the first time, by synthesis, the specificities of these classic amylases long known from their hydrolytic reactions. A rapid approach to equilibrium was found both in maltose synthesis from β-d-glucopyranose by glucoamylase, and in maltotetraose synthesis from β-maltose by β-amylase. Moreover, essentially the same equilibrium (Keq = ca. 0.13) was reached by these homologous hemiacetal-to-secondary carbinol condensations. The configurational inversion accompanying both condensations, finally, reveals their mechanism as one of glycosyl transfer. As reversions represent the only class of carbohydrase-catalyzed reactions not previously shown to follow this mechanism, their addition demonstrates the complete generality of the glycosyl-hydrogen interchange model proposed as the paradigm for the action of the carbohydrases. © 1969.