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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.

SUBMITTER: Bloxham DP

PROVIDER: S-EPMC1162580 | biostudies-other | 1981 Jan

REPOSITORIES: biostudies-other

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Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase.

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Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue.

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The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

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