title: Alanine racemase free energy profiles from global analyses of progress curves.
abstract: Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves. This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism). The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1). steady-state kinetic constants, (2). solvent viscosity dependence, (3). spectral analysis of reaction intermediates, (4). equilibrium overshoots for progress curves measured in D(2)O, and (5). the magnitudes of calculated intrinsic kinetic isotope effects. The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions. At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol.
authors.affiliation: Department of Chemistry and Statistical Laboratory, University of California, One Shields Avenue, Davis, California 95616, USA.
authors.forename: M Ashley
authors.initials: MA
authors.lastname: Spies
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Woodward
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: Watnik
authors.forename: Michael D
authors.initials: MD
authors.lastname: Toney
mesh_terms: D000409:Alanine / Q000737:chemistry*; D000411:Alanine Racemase / Q000032:analysis / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008962:Models, Theoretical; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011809:Quinones / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013237:Stereoisomerism
chemical_list: D011809:Quinones; D012997:Solvents; D003903:Deuterium; D000411:Alanine Racemase; D000409:Alanine