This project involves a continuation of our studies on the enzyme L-aspartase, which catalyzes the reversible addition of ammonia to fumaric acid. The long term objectives of this project are to acquire a detailed understanding, at the molecular level, of the events involved in the mechanism and the regulation of the reaction catalyzed by L-aspartase, and by several other aspartate-utilizing metalloenzymes. The urea cycle enzyme argininosuccinase catalyzes a related reaction involving the addition of arginine to fumaric acid. Partial loss of argininosuccinase activity has been implicated in an inborn error of metabolism in humans associated with mental deficiencies. The bifunctional enzyme aspartokinase- homoserine dehydrogenase also occupies a key position in metabolism, the commitment step to the biosynthesis of four amino acids starting from L-aspartic acid.
The specific aims proposed in this research plan are to: probe the detailed kinetic and chemical mechanism of this enzyme system; establish the role of metal ion activators and inhibitors; explore the dual role of L- aspartic acid as a substrate and an activator for the enzyme; and determine the structure and orientation of the binding sites on L- aspartase. The techniques that will be applied in order to address the specific aims of the research project will include equilibrium dialysis and kinetic binding studies of metal ions, substrate analogs, and activators. A range of kinetic studies, including initial velocity, product inhibition, pH, and isotope effect studies, will be utilized to elucidate dynamic information about the reaction catalyzed by L-aspartase. Magnetic resonance studies, including NMR paramagnetic relaxation studies to examine enzyme-- substrate and enzyme-activator interactions, NMR and EPR studies to examine metal ion interactions, low temperature NMR studies to characterize intermediates in the catalytic cycle, and x-ray crystallographic studies of enzyme-substrate and analog complexes, will be utilized to provide structural information about the interaction of metal ions, substrates and activators with L- aspartase. Taken together these techniques offer the opportunity to conduct a detailed examination of the events involved in the mechanism of L-aspartase and some related enzymes, and to examine the role played by metal ion and substrate effectors for these enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034542-04
Application #
3285759
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-09-01
Project End
1993-01-31
Budget Start
1990-08-01
Budget End
1993-01-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Akron
Department
Type
Schools of Arts and Sciences
DUNS #
City
Akron
State
OH
Country
United States
Zip Code
44325
Viola, R E (2000) L-aspartase: new tricks from an old enzyme. Adv Enzymol Relat Areas Mol Biol 74:295-341
Saribas, A S; Schindler, J F; Viola, R E (1994) Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase. J Biol Chem 269:6313-9
Schindler, J F; Viola, R E (1994) Mechanism-based inactivation of L-aspartase from Escherichia coli. Biochemistry 33:9365-70
Shi, W; Kidd, R; Giorgianni, F et al. (1993) Crystallization and preliminary X-ray studies of L-aspartase from Escherichia coli. J Mol Biol 234:1248-9
Karsten, W E; Viola, R E (1991) Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes. Arch Biochem Biophys 287:60-7
Falzone, C J; Karsten, W E; Conley, J D et al. (1988) L-aspartase from Escherichia coli: substrate specificity and role of divalent metal ions. Biochemistry 27:9089-93
Karsten, W E; Gates, R B; Viola, R E (1986) Kinetic studies of L-aspartase from Escherichia coli: substrate activation. Biochemistry 25:1299-303
Karsten, W E; Hunsley, J R; Viola, R E (1985) Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography. Anal Biochem 147:336-41