Degree Type

Dissertation

Date of Award

2007

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Richard B. Honzatko

Second Advisor

Herbert J. Fromm

Third Advisor

Richard B. Honzatko

Abstract

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.

DOI

https://doi.org/10.31274/rtd-180813-16983

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Daniel Joseph Binkowski

Language

en

Proquest ID

AAI3383365

OCLC Number

491476498

ISBN

9781109454758

File Format

application/pdf

File Size

133 pages

Included in

Biochemistry Commons

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