Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Richard B. Honzatko
Herbert J. Fromm
Richard B. Honzatko
Phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) catalyzes the eighth step of de novo IMP biosynthesis in bacteria, and the seventh step in humans. The SAICAR synthetase reaction is analogous to that of adenylosuccinate synthetase (first committed step of AMP biosynthesis), using ATP to ligate 5-amino-imidazole-4-carboxy ribonucleotide and L-aspartate to produce 5-aminoimidazole-4(N-succinylcarboxamide) ribonucleotide (SAICAR). SAICAR synthetase and other enzymes of purine nucleotide biosynthesis are targets of natural products that impair cell growth. Prior to these studies, the kinetic mechanism of any SAICAR synthetase was unknown. Herein are reported the kinetic mechanisms of bacterial and human SAICAR synthetase activities, the structure of the bacterial enzyme from Escherichia coli, and substrate recognition properties of both the human and bacterial forms of the enzyme. The human enzyme is bifunctional, combining 5-aminoimidazole ribonucleotide (AIR) carboxylase activity with SAICAR synthetase activity. A determination of the kinetic mechanism of SAICAR synthetase requires the absence of AIR carboxylase activity. A slow, tight-binding inhibitor (4-Nitro-5-aminoimidazole ribonucleotide) and the mutation of a residue critical to catalysis independently eliminated interfering AIR carboxylase activity. The kinetic mechanism of the SAICAR synthesis is the same for the two forms of AIR carboxylase-impaired enzyme, but differences in kinetic parameters demonstrate a linkage mechanism between the two types of active site in the human bifunctional enzyme. Human SAICAR synthetase may impose a metering function that insures constant output of SAICAR over a ten-fold variation in the concentration of CAIR.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Daniel Joseph Binkowski
Binkowski, Daniel Joseph, "Kinetic studies of Escherichia coli and human SAICAR synthetase" (2007). Retrospective Theses and Dissertations. 15779.