Probing enzyme-substrate interactions at the catalytic subsite of Leuconostoc mesenteroides sucrose phosphorylase with site-directed mutagenesis: the roles of Asp49 and Arg395

Sucrose phosphorylase is a bacterial alpha-transglucosidase that catalyses glucosyl transfer from sucrose to phosphate, releasing D-fructose and alpha-D-glucose 1-phosphate as the product of the first (enzyme glucosylation) and second (enzyme deglucosylation) step of the enzymatic reaction, respectively. The transferred glucosyl moiety of sucrose is accommodated at the catalytic subsite of the phosphorylase through a network of charged hydrogen bonds whereby a highly conserved residue pair of Asp and Arg points towards the equatorial hydroxyl at C-4. To examine the role of this 'hyperpolar' binding site for the substrate 4-OH, we have mutated Asp(49) and Arg(395) of Leuconostoc mesenteroides sucrose phosphorylase individually to Ala (D49A) and Leu (R395L), respectively, and also prepared an 'uncharged' double mutant harbouring both site-directed substitutions. The efficiency for enzyme glucosylation from sucrose was massively decreased in purified preparations of D49A (10(7)-fold) and R395L (10(5)-fold) as compared to wild-type enzyme. The double mutant was not active above the detection limit. Enzyme deglucosylation to phosphate proceeded relatively efficient in D49A as well as R395L, about 500-fold less than in the wild-type phosphorylase. Substrate inhibition by phosphate and a loss in selectivity for reaction with phosphate as compared to water were new features in the two mutants. Asp(49) and Arg(395) are both essential in the catalytic reaction of L. mesenteroides sucrose phosphorylase.