A detailed investigation of the properties of lactate dehydrogenase  in which the 'Essential' cysteine-165 is modified by thioalkylation.
The reaction of pig heart lactate dehydrogenase with methyl methanethiosulphonate resulted in the modification of one thiol group per protomer, and this was located at cysteine-165 in the enzyme sequence. On reduction, both the thiomethylation of cysteine-165 and any changes in kinetic properties of the enzyme were completely reversed. Cysteine-165 has been considered essential for catalytic activity; however, cysteine-165-thiomethylated dehydrogenase possessed full catalytic activity, although the affinity of the enzyme for carbonyl-or hydroxy-containing substrates was markedly decreased. The nicotinamide nucleotide-binding capacity was unaffected, as judged by the formation of fluorescent complexes with NADH. The enzyme-mediated activation of NAD+, as judged by sulphite addition, was unaffected in thiomethylated lactate dehydrogenase. However, the affinity of oxamate for the enzyme--NADH complex was decreased by 100-fold and it was calculated that this constituted a net increase of 10.4 kJ/mol in the activation energy for binding. Thiomethylated lactate dehydrogenase was able to form an abortive adduct between NAD+ and fluoropyruvate. However, the equilibrium constant for adduct formation between pyruvate and NAD+ was too low to demonstrate this complex at reasonable pyruvate concentrations. A conformational change in the protein structure on selective thiomethylation was revealed by the decreased thermostability of the modified enzyme. The alteration of lactate dehydrogenase catalytic properties on modification depended on the bulk of the reagent used, since thioethylation resulted in an increase in Km for pyruvate (13.5 +/- 3.5 mm) and an 85% decrease in maximum catalytic activity. The implications of all these findings for the catalytic mechanism of lactate dehydrogenase are discussed.
D000478:Alkylation; D001665:Binding Sites; D002087:Butyrates; D003545:Cysteine; D006038:Glyoxylates; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives; D009243:NAD; D011487:Protein Conformation; D011773:Pyruvates; D013886:Thiosulfonic Acids
D002087:Butyrates; D006038:Glyoxylates; D011773:Pyruvates; D013886:Thiosulfonic Acids; D009243:NAD; D008741:Methyl Methanesulfonate; D007770:L-Lactate Dehydrogenase; D003545:Cysteine