A detailed investigation of the properties of lactate dehydrogenase in which the ‘Essential’ cysteine-165 is modified by thioalkylation

Abstract

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.

The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase

“…The substrate analogue, fluoropyruvate, has previously been shown to readily form an adduct with NAD+ at the active site of lactate dehydrogenase (Eismann et al, 1965). The enzyme is particularly sensitive to substrate inhibition by fluoropyruvate, which is consistent with the fact that the equilibrium constant for adduct formation with this compound is about 50 times more favourable than for the corresponding adduct formation with pyruvate (Bloxham et al, 1979). Again, like pyruvate, both the rate of adduct formation and the rate of enolization -varied in an identical manner with the concentration of fluoropyruvate (Table 2) so that the ratio of the two rates remained constant at a value of about 1.3.…”

“…The kinetic isotope effect, kHIkD, is the ratio of the initial rates for pyruvate and previously described a modified pig heart lactate dehydrogenase in which the active-site cysteine is thiomethylated (enzyme-SH -÷ enzyme-S-S-CH3), with the result that the enzyme has a much higher Km for pyruvate (Bloxham & Wilton, 1977). It is impossible to demonstrate adduct formation with pyruvate for this enzyme owing to the unfavourable equilibrium constant; however, the substrate analogue fluoropyruvate did form such an adduct with this modified enzyme (Bloxham et al, 1979) and the rate of formation of adduct was identical with that for the unmodified enzyme.…”

Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

“…As modification of this amino acid inactivates these enzymes, an essential function in catalysis has been proposed for this residue. However, methanethiolated lactate dehydrogenase retains full catalytic activity, but with a large increase in the Km for pyruvate (Bloxham & Wilton, 1977;Bloxham et al, 1979). The thiol is not involved, therefore, in any essential covalent step in the catalytic mechanism, but in the binding of substrate, possibly by stabilizing an ionic interaction.…”

The development of SS′-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase

“…Of these residues cysteine-165 was once considered essential for catalysis (Fondy et al, 1965;Holbrook & Pfleiderer, 1965;Holbrook, 1966), but it is now appreciated that its role is principally in substrate binding and maintenance of the correct enzyme conformation (Bloxham & Wilton, 1977;Bloxham et al, 1979). Whatever the exact role of the thiol it is buried in the protein structure and is not readily accessible to externally added reagents unless they are reasonably non-polar (Holbrook & Pfleiderer, 1965;Kolkenbrock et al, 1978) or alternatively if the protein is denatured (Bloxham et al, 1979). When lactate dehydrogenase was allowed to react with SS'-pentamethylenebis-(methanethiosulphonate) the enzyme was progressively inactivated (Fig.…”