Role of non-covalent enzyme-substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda

Steady-state kinetic studies of the enzymic glucosyl transfer to and from p hosphate catalysed by cellobiose phosphorylase from Cellulomonas uda have s hown that this enzyme operates by a ternary-complex kinetic mechanism in wh ich beta -cellobiose binds before phosphate, and beta -D-glucose and alpha -D-glucopyranosyl phosphate are released in that order. alpha -D-Glucopyran osyl fluoride (but not beta -D-glucopyranosyl fluoride) serves as alternati ve glucosyl donor for beta -cellobiose synthesis with a specificity constan t that is one-ninth that of the corresponding enzymic reaction with alpha - D-glucopyranosyl phosphate (approximate to 20000 M-1 . s(-1) at 30 degreesC ). The kinetic parameters for a complete series of deoxy and deoxyfluoro an alogues of D-glucose have been determined and the data yield estimates of t he net strengths of hydrogen-bonding interactions with the non-reacting hyd roxy groups of D-glucose at the transition state (0.8-4.0 kcal/mol, where 1 cal = 4.184 J) and enable the prediction of the polarities of these hydrog en bonds. Each hydroxy group functions as donor of a hydrogen for bonding t o probably a charged (at 3-OH) or neutral (at 2-OH and 6-OH) acceptor group on the enzyme. The equatorial 1-OH is essential for enzyme activity. Deriv atives of D-glucose in which the 1-OH or the reacting 4-OH were replaced by hydrogen or fluorine have been tested as inhibitors to measure their affin ities for the sugar-binding subsite + 1 (numbered from the bond-cleaving/fo rming site). The data show that hydrogen-bonding interactions between the 1 -OH and 4-OH and charged groups on the enzyme stabilize the groundstate ter nary complex of the enzymic synthesis of beta -cellobiose by 2.3 and 0.4 kc al/mol, respectively, and assist the precise positioning of beta -D-glucose for catalysis.