Stopped-flow kinetic studies on the D-lactate dehydrogenase from megasphera elsdenii

The reaction of oxidized D-lactate dehydrogenase with D-lactate and reduced D-lactate dehydrogenase with pyruvate and oxygen was studied in a stopped-flow spectrophotometer. Oxidized enzyme was reduced in a triphasic manner by D-lactate at a variety of pH values. The fastest phase is thought to represent charge-transfer complex formation and is equal to an increase in long wavelength (510-700 nm) absorbance. The two slower phases seem to be due to flavin reduction. At pH 6 the proportion of the flavin reduction which proceeds by either phase is dependent upon the concentration of D-lactate. The reaction between reduced enzyme and oxygen is a simple second-order reaction. However, the oxidation of reduced D-lactate dehydrogenase by pyruvate is biphasic at pH 6 and monophasic at pH 8. At pH 8 there is a rapid increase in absorbance above 510 nm which is again ascribed to formation of a charge-transfer complex. These results are taken to indicate that D-lactate dehydrogenase may exist in two active conformations. At high pH the enzyme operates mainly through only one form while at low pH both forms participate in catalysis.