title: Mechanistic studies on malate dehydrogenase from Escherichia coli.
abstract: Kinetic studies and chemical modification studies using diethylpyrocarbonate and iodoacetate were performed on malate dehydrogenase isolated from Escherichia coli. Product inhibition experiments indicate that this enzyme follows an ordered Bi Bi kinetic mechanism, similar to other dehydrogenases, while log V/K profiles reveal that one ionizing group with a pKa between 7.8 and 8.7 acts as a general acid/general base in the catalytic mechanism. Log V profiles indicate that malate binds to the correctly protonated form of the enzyme while binding of OAA is pH-independent. Chemical modification experiments implicate an activate site histidine residue is essential for catalytical activity. A primary kinetic isotope effect of 1.44 +/- 0.14 on V/K using malate-2-d at pH 9.0 was measured while no isotope effect was observed on Vmax which is, again, similar to other dehydrogenases. This implies that proton abstraction is partially rate determining under nonsaturating conditions. Within experimental error, small isotope effects were observed on V and V/K (1.219 +/- 0.188 and 1.078 +/- 0.064, respectively) when NADD was utilized indicating that release of cofactor may be rate limiting.
authors.affiliation: Department of Chemistry, Eastern Illinois University, Charleston 61920, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Wright
authors.forename: F J
authors.initials: FJ
authors.lastname: Zhao
authors.forename: J
authors.initials: J
authors.lastname: Rardin
authors.forename: J
authors.initials: J
authors.lastname: Milbrandt
authors.forename: M
authors.initials: M
authors.lastname: Helton
authors.forename: N C
authors.initials: NC
authors.lastname: Furumo
mesh_terms: D001665:Binding Sites; D003903:Deuterium; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009243:NAD / Q000378:metabolism
chemical_list: D007461:Iodoacetates; D009243:NAD; D006639:Histidine; D003903:Deuterium; D008291:Malate Dehydrogenase; D004047:Diethyl Pyrocarbonate; D019807:Iodoacetic Acid