Structural Dissection of the HGPRT Reaction Mechanism

Southern Research Institute, Birmingham AL

Investigators

Abstract

DESCRIPTION (Applicant?s abstract): For the past few years the principal investigator and his group have studied hypoxanthine-guanine phosphoribosyltransferase (HGPRT), a key purine salvage enzyme in Toxoplasma gondii. T. gondii is a pervasive parasitic protozoan that is a leading cause of morbidity and mortality in AIDS patients. The long term research goal is to design HGPRT inhibitors that are useful as drugs to treat toxoplasmosis. The principal investigator?s prior work on HGPRT has provided atomic resolution structural information, mutagenesis data, and enzymological results that together address several key questions regarding the HGPRT reaction mechanism. This work has raised several new mechanistic questions. Additional biochemical and structural information is needed to fully understand HGPRT as an enzyme. This added knowledge will have fundamental impact on our understanding of enzyme reactions generally, and it will also provide a solid foundation for the rational design of drugs that target HGPRT. To provide this fundamental information, the principal investigator proposes to: (1) test four hypotheses on the HGPRT reaction mechanism, regarding cis-trans isomerization of the Leu78-Lys79 active site peptide bond during catalysis, the roles played by active site loops Il and IIT, and the basis for selective xanthine use by T. gondii HGPRT; (2) complete the determination of additional T. gondii HGPRT crystal structures, using x-ray diffraction data sets already in hand (wildtype and mutants with a variety of ligands); (3) solve new structures of HGPRT bound to transition state analogues and other existing compounds; and (4) determine whether HGPRT heterotetramers or only homotetramers exist in T. gondii, and to compare the biochemical and enzymological properties of the HGPRT isozymes. Three collaborators will assist the principal investigator in specific, limited portions of this research. Prof. David Roos will transfect our HGPRT isozymes into T. gondii and return parasite lysates to the applicant?s laboratory for analysis. Prof. Charles Grubmeyer will perform single-turnover kinetics studies of wildtype and mutant T. gondii HGPRTs. Prof. Herbert Cheung will consult on fluorescence studies.