title: Analysis of wild-type and mutant aspartate aminotransferases using integrated rate equations.
abstract: A general integrated rate equation was fit to reaction progress curves catalyzed by wild-type E. coli aspartate aminotransferase and the site-specific mutant enzymes, H193Q and Y70F. A nonlinear step-regression code, revised for this study selected from all kinetic constants in a general integrated rate equation for all unbranched enzyme mechanisms with stoichiometries upto two substrates and two products including terms for substrate inhibitions and that of an exogenous inhibitor. For each aspartate aminotransferase enzyme studied only kinetic constants consistent with a substituted enzyme mechanism were found statistically significant, thus the enzyme mechanism and sources of inhibition were determined objectively by statistics. The kinetic constants for wild-type and Y70F aspartate aminotransferase were similar to those previously reported indicating the validity of the integrated rate equation analysis. Minor changes in kinetic constants were observed for the H193Q mutant enzyme suggesting that the catalytic effects of the electrostatic hydrogen bonding network extending from the pyridine nitrogen of the cofactor through Asp-222, His-189 ends prior to His-193.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA. schiller@welchlink.welch.jhu.edu
authors.forename: M R
authors.initials: MR
authors.lastname: Schiller
authors.forename: L D
authors.initials: LD
authors.lastname: Holmes
authors.forename: E A
authors.initials: EA
authors.lastname: Boeker
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D003998:Dicarboxylic Acids / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D009154:Mutation; D012044:Regression Analysis
chemical_list: D000600:Amino Acids, Dicarboxylic; D003998:Dicarboxylic Acids; D001219:Aspartate Aminotransferases