Kinetic studies of Escherichia coli and human SAICAR synthetase

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2007-01-01
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Binkowski, Daniel
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Richard B. Honzatko
Herbert J. Fromm
Richard B. Honzatko
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Biochemistry, Biophysics and Molecular Biology

The Department of Biochemistry, Biophysics, and Molecular Biology was founded to give students an understanding of life principles through the understanding of chemical and physical principles. Among these principles are frontiers of biotechnology such as metabolic networking, the structure of hormones and proteins, genomics, and the like.

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The Department of Biochemistry and Biophysics was founded in 1959, and was administered by the College of Sciences and Humanities (later, College of Liberal Arts & Sciences). In 1979 it became co-administered by the Department of Agriculture (later, College of Agriculture and Life Sciences). In 1998 its name changed to the Department of Biochemistry, Biophysics, and Molecular Biology.

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1959–present

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  • Department of Biochemistry and Biophysics (1959–1998)

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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.

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Mon Jan 01 00:00:00 UTC 2007