This research will concentrate on the understanding of the molecular basis of enzyme action. In particular, to study the actual protein molecules involved in the regulatory process and to examine the molecular basis by which an enzyme can regulate its own activity and how the cell can regulate the biosynthesis of that particular enzyme. The understanding of cellular regulation would have a great impact on our grasp of cellular differentiation and as a consequence of this cures for cancer and birth defects may be found. Emphasis will be directed towards two aspects of control of the pyrimidine biosynthesis pathway, the products of which are necessary for DNA replication. First, the enzyme aspartate transcarbamylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms; and second, the pyrS gene, a new gene which we recently discovered, which is directly involved in control of this pathway. In addition, we will use E. coli alkaline phosphatase as a model to study the in vitro modification of enzyme activity. After the analysis of our current set of over 200 mutant versions of aspartate transcarbamylase with single amino acid substitutions is completed, we will generate specific mutants by in vitro mutagenesis to acquire sufficient information to propose a mechanism for the homotropic and heterotropic interactions of the enzyme. After the genetic characterization of the pyrS gene is completed, we will further characterize the pyrS gene-product. We will also investigate the interaction of the pyrS gene-product with its metabolic effector and its DNA binding site, in order to determine the mechanism by which it exerts control over the pyrimidine pathway. Finally, will be used in vitro mutagenesis to delineate the catalytic mechanism and cooperativity of alkaline phosphatase and to use these techniques to alter the specificity and catalytic efficiency of the enzyme. The use of recombinant DNA technology will substantially increase our ability to answer these fundamental questions and, therefore, the specific aim of this Research Career Development Award is to provide me an opportunity to integrate this new technology into my research.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Modified Research Career Development Award (K04)
Project #
5K04DK001429-05
Application #
3072403
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-09-01
Project End
1990-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Boston College
Department
Type
Schools of Arts and Sciences
DUNS #
045896339
City
Chestnut Hill
State
MA
Country
United States
Zip Code
02467
Xu, X; Kantrowitz, E R (1991) A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity. Biochemistry 30:7789-96
Tauc, P; Vachette, P; Middleton, S A et al. (1990) Structural consequences of the replacement of Glu239 by Gln in the catalytic chain of Escherichia coli aspartate transcarbamylase. J Mol Biol 214:327-35
Stebbins, J W; Zhang, Y; Kantrowitz, E R (1990) Importance of residues Arg-167 and Gln-231 in both the allosteric and catalytic mechanisms of Escherichia coli aspartate transcarbamoylase. Biochemistry 29:3821-7
Kantrowitz, E R; Lipscomb, W N (1990) Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition. Trends Biochem Sci 15:53-9
Dembowski, N J; Newton, C J; Kantrowitz, E R (1990) Function of serine-171 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamoylase. Biochemistry 29:3716-23
Newton, C J; Kantrowitz, E R (1990) Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase. Biochemistry 29:1444-51
Newton, C J; Kantrowitz, E R (1990) The regulatory subunit of Escherichia coli aspartate carbamoyltransferase may influence homotropic cooperativity and heterotropic interactions by a direct interaction with the loop containing residues 230-245 of the catalytic chain. Proc Natl Acad Sci U S A 87:2309-13
Chaidaroglou, A; Kantrowitz, E R (1989) Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity. Protein Eng 3:127-32
Stebbins, J W; Kantrowitz, E R (1989) The importance of the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain for the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase. J Biol Chem 264:14860-4
Hsuanyu, Y; Wedler, F C; Kantrowitz, E R et al. (1989) Site-specific mutation of Tyr240----Phe in the catalytic chain of Escherichia coli aspartate transcarbamylase. Consequences for kinetic mechanism. J Biol Chem 264:17259-65

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