STRUCTURE/FUNCTION RELATIONSHIPS OF HPPK

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The goal of the proposed multidisciplinary research is to elucidate the structure-function relationships of 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase (HPPK), the first enzyme in the folate biosynthesis pathway. Our system is recombinant E. coli HPPK because the E. coli isozyme is the best model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. The results will provide the detailed information on the structure of HPPK and how it catalyzes the transfer of pyrophosphate. The proposed research is of fundamental importance to enzymology because very little is known about the structures and mechanisms of any pyrophosphokinases. Furthermore, the structural and mechanistic information that will be obtained from the proposed research will be essential for design of inhibitors for HPPK. Since most microorganisms must synthesize folate de novo but man and animals cannot and don't need to, HPPK inhibitors may become new antimicrobial agents. This is of great biomedical significance especially in light of rapidly increasing antibiotic resistance in recent years that has rendered the current antibiotics ineffective for treating many microbial infections and caused a worldwide health care crisis. In Specific Aim l, the kinetic pathway and energetics of the HPPK-catalyzed reaction will be determined by steady-state kinetic, transient kinetic, thermodynamic and stereochemical analyses. In Specific Aim 2, site-directed mutagenesis, in conjunction with biochemical and biophysical characterizations of the mutants, will be used to identify the active-site residues and to elucidate their roles in substrate binding and catalysis. In Specific Aim 3, biophysical methods will be used to assess the effects of mutations on the structure and stability of HPPK at both free and substrate-bound states. In Specific Aim 4, the solution structure of the free HPPK will be determined by double-and triple-resonance multidimensional NMR. In specific Aim 5, NMR will be used to identify the active-site residues and to determine the structures of HPPK in complex with substrate analogues.