title: The chemical reactivity of the histidine-195 residue in lactate dehydrogenase thiomethylated at the cysteine-165 residue.
abstract: The specific thiomethylation of cysteine-165 (insertion of a methylthio group, CH3-S-) in pig heart lactate dehydrogenase results in a decreased affinity for carbonyl ligands that is accompanied by a decreased nucleophilic reaction of histidine-195 with diethyl pyrocarbonate. The rate constants at 10 degrees C for the modification of native and thiomethylated lactate dehydrogenase by diethyl pyrocarbonate were 173 M-1 . s-1 and 8.7 M-1 . s-1 respectively. It was found that 0.86 +/- 0.07 histidine residue per subunit reacted with diethyl pyrocarbonate in thiomethylated lactate dehydrogenase. This reaction was not affected in the enzyme-NADH binary complex, but was diminished in the enzyme-NADH-oxamate ternary complex. In the enzyme-NADH complex the reaction of diethyl pyrocarbonate was controlled by two groups with pKa 6.8 and 7.9. The decreased reactivity of histidine-195 was selective in thiomethylated lactate dehydrogenase, since the reactivity of arginine and/or lysine residues was enhanced.
mesh_terms: D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors*; D008024:Ligands; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives; D011773:Pyruvates / Q000494:pharmacology; D013440:Sulfides
chemical_list: D005561:Formates; D008024:Ligands; D011773:Pyruvates; D013440:Sulfides; C014674:methyl methanethiosulfonate; D006639:Histidine; C017092:bromopyruvate; D008741:Methyl Methanesulfonate; D007770:L-Lactate Dehydrogenase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate