The proposed research is a continuation of the study of the properties and mechanism of action of glucansucrases, Leuconostoc mesenteroides B- 512F dextransucrase, Streptococcus mutans 6715 dextransucrase (glucosyltransferase-soluble [GTF-S]) and S. mutans mutansucrase (glucosyltransferase-insoluble [GTF-I]). The study will focus on five aspects: (1) The study of the sucrose binding specificity. Sucrose analogues with the hydroxyl group at C-1', -6', and -2 replaced by H, F, and NH2 will be synthesized and studied as inhibitors or substrates for dextran synthesis. (2) The study of acceptor binding specificity. Analogues of alpha-methyl-D-glucopyranoside and 1,5-anhydro-maltitol in which the C-6-OH is replaced by H, F, and NH2 and alpha-Me-Glc in which the OH at C-2, -3, and -4 are replaced by H or inverted will be synthesized and studied as inhibitors of glucan synthesis and the structures of any resulting acceptor products determined. (3) The synthesis and study of 1-alpha-(haloacetyl)-D-glucopyranosides as specific active-site-directed inhibitors that alkylate the catalytic imidazole groups. (4) The study of the effect of acceptor reactions on dextran synthesis and the formation of branch linkages. Studies will be conducted on (a) the effect of the concentration of maltose; (b) the effect of viscosity on the structure of dextran synthesis; (c) the minimum size of isomaltodextrin required for the formation of a branch linkage; and (d) the reaction of GTF-I and GTF-S with maltodextrins DP 8-12 and with starch. (5) Study of the active site and the mechanism of action. Three experiments are proposed: (a) the determination of the number of sucrose binding-sites per enzyme molecule by equilibrium dialysis and equilibrium gel-filtration using [14C]-6-deoxy sucrose; (b) the determination of the number of acceptor binding-sites using the same techniques with [3H]-alpha-methyl-D-glucopyranoside and with [14C]maltose; and (c) the determination of the ratio of the acceptor reaction to the glycan synthesis as a function of partial inactivation by diethylpyrocarbonate and by dye photo-oxidation. The health significance involves a better understanding of the mechanism of glucansucrases and the formation of dental plaque by understanding: the substrate and acceptor binding specificity and thereby the development of inhibitors and the effect of acceptors and viscosity on dextran and plaque structure.
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