Mammalian d-2-hydroxy acid dehydrogenase. Effect of inhibitors and reaction sequence

1. The reaction of d-2-hydroxy acid dehydrogenase with d-lactate and 2,6-dichlorophenol-indophenol (DCIP) at pH8.6 yields reciprocal plots of 1/rate versus 1/d-lactate], at different DCIP concentrations, which appear to be parallel. However, at pH7.55, or in the presence of the competitive inhibitor oxalate at pH8.6, the plots are convergent. This is inconsistent with the mechanism previously proposed for this enzyme. 2. The pattern of inhibition by the product, pyruvate, is consistent with either an Ordered mechanism or an Iso Theorell-Chance mechanism. 3. The observation that the enzyme forms a complex with d-lactate favours the Ordered reaction. In this, first d-lactate and then DCIP bind to the enzyme to form a ternary complex, from which pyruvate and reduced DCIP dissociate in that order.