This proposal describes a comprehensive series of experiments designed to elucidate the structure and mechanism of glycosyltranferases from S. mutans (and other bacteria) in the context of the role of these enzymes in initiating dental caries of smooth enamel surfaces.
One specific aim i s to determine the amino acid sequence of a peptide at the active-site of three S. mutans glycosyltransferases: (1) Alpha-1,6-D-glucan synthetase (dextransucrase); (2) Alpha-1,3-D-glucan synthetase (mutansucrase); and (3) fructosyltransferase; and the Alpha-1,6-D-glucan synthetase from S. sanguis and L. mesenteroides. The enzyme active-site can be labeled with radioactive glucose by trapping, under acidic conditions, a glucosyl-enzyme covalent intermediate. Protein cleavage agents that are effective at low pH will be used to hydrolyze the enzyme into peptide fragments that will then be separated by reverse-phase high-performance liquid chromatography. The amino acid composition of glucosyl-peptides will be determined, and the peptides will be sequenced using a gas-phase protein sequenator. The sequences of the active-site peptide from each enzyme will be compared and analyzed for homologies with other proteins catalogued in the Protein Sequence Database.
A second aim i s the structural comparison of glucosyltransferases from S. mutans serotypes a to g, S. sanguis, and L. mesenteroides. Glucosyltrans ferases (Alpha-1,6-D-glucan synthetase, Alpha-1,3-D-glucan synthetase, highly-branched Alpha-1,6-D-glucan synthetase, and basic Alpha-1,6-D-glucan synthetase) will be purified by affinity chromatography followed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzyme will be hydrolyzed using enzymatic and/or chemical cleavage procedures, then chromatographed by high-performance liquid chromatography. The peptide elution patterns will be analyzed for common structural domains among the enzymes. In addition, the N-terminal amino acid sequence of each enzyme will be determined as a second source of protein structure comparison.
The third aim of the project is further analysis of the catalytic mechanisms of glycosyltransferases. Sucrose will be prepared with 18O as the glycosidic oxygen to determine the degree at which glycoside bond hydrolysis is rate limiting and to determine the affect of glycosyl acceptors on the rate of bond hydrolysis; and the kinetic mechanisms of S. mutans mutansucrase and fructosyltransferase will be established to assess the catalytic similarity of these and other well-characterized glycosyltransferases.
