The application is submitted under the Dentist Scientist Award program and outlines a combined curriculum leading to a PhD in Craniofacial Biology and a Certificate in the specialty of Advanced Periodontics. The course-work of the curriculum spans a five-year program with basic science courses for both the doctorate and periodontics certificate completed in the first year an Periodontics clinically-related courses and the clinical practice are spread through years 2 and 5 but are emphasized in years 4 and 5. Laboratory research is present each semester for 5 years until completion of the award program. The major thrust of the thesis research begins in year 2. The research project is a modern protein chemistry and enzymology study of the enzyme fructosyltransferase from Streptococcus mutans. Fructosyltransferases have been only moderately studied even though they are major products of two important oral bacteria, S. mutans and Actinomyces viscosus. The research proposal focuses on an approach to unambiguously identify a peptide segment that contains an active-site amino acid directly involved catalysis. The process is dependent on trapping a fructosyltransferase fructosyl-enzyme covalent complex. As part of the research, enzyme kinetic experiments will be performed to determine the relative steady-state abundance of a fructosyl-enzyme intermediate as an estimate of the anticipated yield of the trapped complex. In the process the kinetic studies will provide the first insight into the enzyme mechanism. If the enzyme behaves similar to the more thoroughly studied enzyme, levansucrase from Bacillus subtillis (which has substantial sequence homology with S. mutans fructosyl-transferase), it will be possible to trap a radiolabeled fructosyl-enzyme complex by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The covalent complex will be proteolytically digested, and a fructosyl peptide isolated and sequenced. Since the fructosyl bond will likely be very labile and cleaved during sequence analysis, the specific amino acid linked to fructose may require methods such as mass spectrometry fragmentation analysis or on-line fast-atom bombardment mass spectrometry with N- or C-terminal proteolysis. In the final phase of the research, site-directed mutagenesis will be performed on the previously cloned and sequenced ftf gene to confirm identification of the catalytic amino acid and possibly discover additional details of the reaction mechanism.
