Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
SAICAR synthetase catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis: ATP + L-aspartate + CAIR → ADP + Pi + SAICAR Enzymes involved in nucleotide synthesis are common targets for chemotherapeutic and antimicrobial drugs. They work by reducing the rate of growth of rapidly proliferating cells, thus preventing tumor growth and microbial fecundity. L-Alanosine, an L-aspartate analogue that acts as a substrate for SAICAR synthetase, is a natural product with antiviral and antitumor activities. The product of the reaction is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase and is responsible for L-alanosine toxicity. L-Alanosine may be effective as a chemotherapeutic agent in combination with other drugs for certain T-Cell lymphocyte tumors.;Crystal structures of E. coli SAICAR synthetase with CAIR revealed two new Mg2+ binding sites. The binding of CAIR and metals orders a loop (residues 35-39). Site-directed mutagenesis experiments and crystal structures reveal a role in L-aspartate binding for this loop. Analogs of CAIR (AICAR and IMP) are phosphorylated, as revealed by crystal structures, and likely mimic a putative phosphoryl intermediate of the substrate. Positional Isotope Exchange (PIX) experiments show migration of 18O from the bridging position of gamma-18O-ATP to terminal positions of the beta-phosphoryl group only in the presence of CAIR, indicative of the formation of a phosphoryl intermediate.;The enzyme from humans combines SAICAR synthetase and AIR carboxylase activities. The kinetic mechanism of human SAICAR synthetase was Steady State Ordered (CAIR first, ATP second, and L-aspartate last). Elimination of AIR carboxylase activity by mutation or by direct inhibition causes a 10-fold difference in the "on" rate constant for CAIR, suggesting a functional linkage between the AIR carboxylase and SAICAR synthetase active sites.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Nathaniel Daum Ginder
Ginder, Nathaniel Daum, "The structure and mechanism of SAICAR synthetase" (2008). Retrospective Theses and Dissertations. 15798.