Studies on the Mechanism and Control of Enzyme Action

Iowa State University, Ames IA

Investigators

Abstract

9985565 Fromm Adenylosuccinate synthetase, a ubiquitous enzyme, participates in two important metabolic pathways, the purine nucleotide cycle and the de novo biosynthesis of adenine nucleotides. The former maintains ATP levels in active muscle tissue, whereas the latter provides essential precursors for RNA and DNA biosynthesis. Even in organisms that lack a pathway for de novo purine biosynthesis, adenylosuccinate synthetase still plays a central role in salvage mechanisms for adenine nucleotides. This research seeks a detailed understanding of the structure-function relationships of the synthetases from Escherichia coli and from mouse. Of central interest is the monomer-dimer subunit equilibrium of adenylosuccinate synthetases and its effect on enzyme activity. Does IMP, one of the substrates of the synthetase, induce a transition from inactive monomers to active synthetase dimers, and if so, is this phenomenon a universal mechanism of regulation for all synthetases? In addition, the mechanism by which stringent effectors influence activity of the E. coli enzyme is unsettled. The stringent effect is a response by E. coli to conditions of stress, such as starvation, resulting in elevated levels of guanine nucleotides, which putatively inhibit adenylosuccinate synthetase and many other enzymes. Is inhibition by some stringent effectors enhanced by the chemical action of adenylosuccinate synthetase, and do stringent effectors inhibit the synthetase by a single mechanism? The synthetase-catalyzed reaction is second order with respect to the essential metal cation, yet in crystal structures only one Mg2+ is present, associated with GDP. The second Mg2+ binds putatively to L-aspartate. Experiments will be designed to test whether the a-carboxyl group of L-aspartate is an essential recognition element for the binding of the second Mg2+. The synthetase catalyzes its overall reaction as a sequence of two partial reactions. Experiments will determine whether the synthesis of 6-phosphoryl-IMP (first reaction) or the synthesis of adenylosuccinate (second reaction) is rate limiting. The proposed research will employ a variety of techniques in physical biochemistry, including X-ray crystallography, 31P NMR, analytical ultracentrifugation, and kinetics. The studies will employ recombinant E. coli and/or mouse synthetases, expressed in and isolated from E. coli. Specific mutants of the E. coli and mouse synthetases will be constructed in order to test several structure-function hypotheses.