Distinction between NAD- and NADH-binding forms of mitochondrial malate dehydrogenase as shown by inhibition with thenoyltrifuoroacetone.
The inhibition of mitochondrial malate dehydrogenase (L-malate : NADH oxidoreductase, EC 1.1.1.37) by 2-thenoyltrifluoroacetone (TTFA) was investigated at pH 8.0 where both forward and backward reactions can be measured. The inhibition with respect to malate is non-competitive at finite NAD concentrations. Increasing the NAD concentrations lowers the slope of the double reciprocal plot so that at infinite NAD the inhibition is uncompetitive. The inhibition with respect to oxaloacetate is non-competitive. Increasing the NADH concentration lowers the slope and intercept of the double reciprocal plot so that at infinite NADH the inhibition is nil. The inhibition with respect to NADH is competitive, whatever the oxaloacetate concentrations are. The inhibition with respect to NAD, at all malate concentrations, is non-competitive. This pattern of inhibition is incompatible with any model assuming that NAD and NADH reacts with identical forms of the enzyme. On the other hand the reciprocating compulsory ordered mechanism, where the two subunits of the dimeric enzyme are working in concert, can account for all the experimental results. It is concluded that NAD and NADH bind to different forms of the enzyme separated by reversible steps. Only one form (see text), the one which binds NADH, can react to form the dead end complex (see text). The similarity between mechanism of inhibition by thenoyltrifluoroacetone and other hydrophobic inhibitors of malate dehydrogenase is discussed.
D001667:Binding, Competitive; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*
D008293:Malates; D013876:Thiophenes; D009243:NAD; D013804:Thenoyltrifluoroacetone; D008291:Malate Dehydrogenase