The chemical reactivity of the histidine-195 residue in lactate dehydrogenase thiomethylated at the cysteine-165 residue

• Published in: Biochemical journal Vol. 193; no. 1; pp. 93 - 97
• Main Author: Bloxham, D P
• Format: Journal Article
• Language: English
• Published: England 01-01-1981
• Subjects: Binding Sites; Chemical Phenomena; Chemistry; Cysteine; Diethyl Pyrocarbonate - pharmacology; Formates - pharmacology; Histidine; Hydrogen-Ion Concentration; Kinetics; L-Lactate Dehydrogenase - antagonists & inhibitors; Ligands; Methyl Methanesulfonate - analogs & derivatives; Pyruvates - pharmacology; Sulfides
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj1930093

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.