Modification of pig heart lactate dehydrogenase with methyl methanethiosulphonate to produce an enzyme with altered catalytic activity.
Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004229:Dithiothreitol / Q000494:pharmacology; D004793:Enzyme Reactivators; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology*; D009206:Myocardium / Q000201:enzymology*; D011773:Pyruvates / Q000378:metabolism; D013552:Swine; D013886:Thiosulfonic Acids / Q000494:pharmacology; D013997:Time Factors
D004793:Enzyme Reactivators; D008698:Mesylates; D011773:Pyruvates; D013886:Thiosulfonic Acids; D008741:Methyl Methanesulfonate; D007770:L-Lactate Dehydrogenase; D004229:Dithiothreitol