The proposal outlines a broad range of experiments concerned with the structure and mechanism of oral streptococcal glucosyltransferases. This family of enzymes plays a central role in supragingival dental plaque formation and ensuing oral diseases such as dental caries. The proposed research builds on progress from the previous project period in which details were advanced on the two fundamental units of the enzyme -the catalytic site and the glucan-binding domain. For the catalytic site, a peptide containing an aspartic acid that binds glucose as an intermediate in glucosyl transfer from sucrose to an acceptor was isolated, sequenced and found to be homologous with peptides from a wide range of glucosidases and glucosyltransferases crossing evolutionary boundaries from prokaryotes to eukaryotes and plants to animals. The other major segment of the enzyme, the glucan-binding domain, has been proteolytically excised and subjected to studies on its function, size and location within the enzyme primary structure. Three overlapping Specific Aims are developed for the next project period: further research is proposed on the structure of the enzyme catalytic site and glucan-binding domain. S. mutans fructosyltransferase (FTF) does not contain a peptide segment analogous to the glucosyltransferases (GTF) indicating the two enzymes arise from distinct gene sources; the relevant FTF active-site peptide will be labeled and analyzed. The structure of a GTF peptide containing a possible catalytically significant histidine will also be investigated. For the glucan-binding domain, the structure of surface peptides that bind glucan will be labeled, isolated and sequenced; this data will be coordinated with related studies that explore the possibility that the glucan-binding domain is assembled from smaller glucan-binding subdomains. Second, several experiments are concerned with the GTF mechanism: Interaction between the catalytic and glucan-binding domain will be studied in the context of influence of domain interaction on catalysis. Also studied will be the glucose analog D-glucal which is a slow-reacting substrate and powerful inhibitor of many glucosidases and transferases; it may provide clues about reaction intermediates. Third, some novel approaches to inhibit GTF will be examined. Deoxynojirimycin is already known to inhibit the enzyme; for some glucosidases, N-alkyl derivatives of deoxynojirimycin are substantially more potent and will be synthesized and tested on GTF. In addition, experiments have been in progress and will continue in a search for peptide inhibitors of the enzyme. Methods have been developed to generate large numbers of peptides and rapidly screen them for GTF and FTF inhibition. Finally, antibodies against catalytically significant and conserved active-site peptides will be prepared and examined for inhibition and cross reaction among glucosyltransferases.
