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.
