This research is oriented toward understanding the development of the integrated regulatory logic involved in metabolic pathways that require consistent, yet environmentally-responsive, activity. The two most common, basic patterns of metabolic regulation involve the control of gene expression and the biochemical modulation of the activity of enzymes which have already been produced in the cell. The classic interaction of these two systems is being examined in the control of the interrelationship of pyrimidine and arginine metabolism in Escherichia coli. The pivotal enzyme in the control of these essential biosynthetic pathways is aspartate transcarbamoylase (ATCase) and it has a modulating role in the metabolic flux of carbamoyl phosphate into the de novo biosynthesis of either arginine or pyrimidine nucleotides. While the E. coli system provides the most sophisticated model of integrated controls, another pattern of regulatory importance develops in an evolutionary mode in which the individual enzymatic steps become assimilated into a multifunctional biochemical steps within a compact architectural unit. While concentrating on the structural organization and regulation of the pyrLBIX operon in E. coli, this research program contrasts this system with the divergent structural organization and related regulatory changes observed in higher organisms (yeast, myxamoeba, plants and hamster) in order to further an understanding of the allosteric regulation of aspartate transcarbamoylase since extensive chemical, mutational and crystallographic data are available. Furthermore, it is possible to form active hybrid gene systems by exchanging discrete genetic cassettes from functionally and structurally divergent enzymes systems involving or related to the E coli enzyme. These studies have indicated that there are conserved genetic modules which can be rearranged in patterns consistent with aa """"""""modular evolution by cassette shuffling"""""""". There are four overlapping genetic mechanisms which affect the expression of the pyrLBIX operon (attenuation, translational blocking, native promoter access, and RNA polymerase response to endogenous nucleotide pools. In addition, the pyrX cistron encodes a protein which is coordinately expressed with the cistrons for the regulatory and catalytic polypeptides of ATCase. The purpose of this protein is unknown but it has been purified and has immunological cross-identity with some monoclonal antibodies against the ATCase catalytic chain. The mechanistic details of this system will be examined and the physiological significance of these controls will be integrated into a detailed understanding of the metabolic flux through the key regulating enzymes for pyrimidine and arginine biosynthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033191-08
Application #
3282592
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-09-28
Project End
1994-08-31
Budget Start
1992-09-01
Budget End
1993-08-31
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Texas Agrilife Research
Department
Type
Schools of Earth Sciences/Natur
DUNS #
110521739
City
College Station
State
TX
Country
United States
Zip Code
77843
Rastogi, V K; Swanson, R; Hartberg, Y M et al. (1998) Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli. Arch Biochem Biophys 354:215-24
Liu, L; Wales, M E; Wild, J R (1997) Conversion of the allosteric regulatory patterns of aspartate transcarbamoylase by exchange of a single beta-strand between diverged regulatory chains. Biochemistry 36:3126-32
Cunin, R; Wales, M E; Van Vliet, F et al. (1996) Allosteric regulation in a family of enterobacterial aspartate transcarbamylases: intramolecular transmission of regulatory signals in chimeric enzymes. J Mol Biol 262:258-69
Strang, C J; Wales, M E; Brown, D M et al. (1993) Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea. Biochemistry 32:4156-67
Wales, M E; Wild, J R (1991) Analysis of structure-function relationships by formation of chimeric enzymes produced by gene fusion. Methods Enzymol 202:687-706
Beck, D; Kedzie, K M; Wild, J R (1989) Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli. J Biol Chem 264:16629-37
Wales, M E; Mann-Dean, M G; Wild, J R (1989) Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum. Can J Microbiol 35:432-8
Major Jr, J G; Wales, M E; Houghton, J E et al. (1989) Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J Mol Evol 28:442-50
Wales, M E; Hoover, T A; Wild, J R (1988) Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme. J Biol Chem 263:6109-14