; R o o t E n t r y F l > @ C o m p O b j b W o r d D o c u m e n t ! O b j e c t P o o l l > l > 4 @ F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT jack cohen Rhonda Young @ @J 9 @ e = e ! C v x x x & ! T k! * v v b 9501866 Horenstein Significant efforts are being made by the PI to help develop a new biochemistry curriculum at the undergraduate and graduate levels, and to develop an interdisciplinary research program which includes undergraduate participation. The PI has instituted and is further developing a new graduate level course entitled "Enzymes and Enzyme Mechanisms". Over the period of the CAREER award, the PI will use this course and pedagogies as a laboratory for dhe creation of a new textbook on the same topic. Additional planned course development and teaching activities include teaching a special topics course concerned with the use of isotopes in biochemical research, and teaching the senior level Biochemistry course. In relationship to this latter course, the Biochemistry Division is currently investigating the creation of an electronic study guide in Biochemistry, operating over the internet with a local World Wide Web client. The study guide would enable students to access a vast amount of material that could not be presented in traditional printed format. The PI is a co-investigator in his departments renewal application of its previous and highly successful NSF-REU award. His laboratory has sponsored an NSF-REU fellow among odher undergraduate researchers. The PI's undergraduate research commitment is strong. In the first year of his appointment, a total of four undergraduates have worked on original and interdisciplinary research projects. Two have chosen to continue on, and three of the four have represented women and minorities. Undergraduate participation in the PI's research program is and will remain a priority. The research component of this proposal presents a plan for the mechanistic characterization of rat-liver sialyltransferases utilizing steady-state kinetics and multiple kinetic isotope effect experiments. Sialyltransferases (ST's) catalyze the transfer of N acetylneuraminic acid(NeuAc), (or sialic acid), as its cytidine monophosphate conjugate(CMP-NeuAc), to an acceptor hydroxyl group typically at the terminus of an oligosaccharide chain of a glycoprotein or glycolipid. The resultant sialoglycoconjugates are recognition elements in a variety of processes including viral replication, inflammatory responses, and adhesion of metastatic tumor cells. Sialyltransferases have not yet been characterized in mechanistic detail, nor have selective inhibitors been reported. These compounds will further illuminate the mechanism of sialyltransferases. The ST substrate CMP-NeuAc is an unusual one for a glycosyltransferase because it is a sugar-mononucleotide and the anomeric carbon of N-acetylneuraminic acid has an adjacent carboxyl residue. The interplay of these groups in glycosyl transfer is unkno wn and will be investigated in the course of the mechanistic investigation. Steady-state kinetic techniques, kinetic isotope effect experiments, and computational approaches will be used to define the kinetic mechanisms and transition state structure for rat liver a(2~6) and a(2~3) sialyl transferases. Parallel to the above investigations, ST's will be cloned, and overexpressed in yeast.. Current understanding of the mechanisms of glycosyltransferases, and especially sialyltransferases is limited. The availability of transition state analogue inhibitors for sialyltransferases would facilitate mechanistic studies, and perhaps allow for manipulation of glycosylation patterns in vivo. %%% The research program will involve determination of the chemical mechanism of enzymes called sialyltransferases. Sialyltransferases link a type of sugar, sialic acid, to carbohydrate chains of glycoproteins, often found on cell surfaces. In the longer term, the mechanistic information obtained will be used to design and synthesize inhibitors of these enzymes. Sialyltransferase inhibitors may be useful tools to elucidate the biological functions of sialic acid-containing glycoproteins, which are involved in inflammation and metastasis. The education component involves the development of a biochemistry curriculum in the Chemistry Department at the University of Florida. One new course at the undergraduate level is "Biochemistry Laboratory", in which students learn how to create and analyze recombinant DNA molecules using state-of-the-art techniques. Students then use the recombinant DNA to produce a human enzyme, which will be purified and analyzed. Undergraduate students are also involved in research in the Principal Investigators laboratory, providing them with actual research training. New graduate level courses include "Enzymes and Enzyme Mechanisms" in which the fundamentals of enzyme m
