The proposed research is the study of the nature of the active sites of four different but related glucansucrases (glucosyltransferases) from Leuconostoc mesenteroides and Streptococcus mutans strains that synthesize four different but structurally related glucans from sucrose and the correlation of the nature of the active sites with the mechanism of synthesis and inhibition of the glucans. The Streptococcus glucans are of two types, water-insoluble and water-soluble and are directly involved in dental plaque formation and dental caries. The Leuconostoc glucans are related in structural type and properties with the Streptococcus glucans and serve as models for the Streptococcus systems and as important modifiers when added to the Streptococcus systems. The research is organized around three areas: (l) the determination of the nature of the nucleophilic groups at the active site that form covalent intermediates with glucose and glucan during synthesis of the glucan, and the nature of a set of proton exchange groups required for the cleavage of the sucrose glycosidic bond and the formation of the glucan glycosidic bond; (2) the determination of the size of the active enzyme unit and the number of nucleophilic groups and proton exchange groups per active unit; and (3) the study of the mechanism of glucan synthesis and inhibition by (a) the use of chemically synthesized sucrose analogues, specifically modified at either positions 3 or 6 of the glucose moiety, (b) the study of active site directed reagents, synthesized from p-nitrophenyl-Alpha-D-glucoside, (c) the determination of the amount of branching by S. mutans glucosyltransferase-S as a function of various reaction parameters such as pH, temperature, sucrose concentration, time of reaction, ionic strength, and varying concentrations of low molecular weight acceptors, and (d) the determination of the number of glucose sub-binding sites at the active site by studying the kinetics of various sized isomaltodextrins undergoing disproportionation reactions.
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