The isolation and characterization of malate-lactate transhydrogenase from Micrococcus lactilyticus.

The malate-lactate transhydrogenase of Micrococcus lactilyticus has been purified and found to be a colorless protein with a molecular weight of approximately 99,000. The enzyme catalyzes the transfer of hydrogen from 3and 4carbon straight chain L-cu-hydroxy acids to cY-keto acids. The reactions catalyzed are readily reversible and the K,, (L-malate) (pyruvate)/(oxalacetate) (L-lactate) = 1.8. The enzyme tightly contains bound pyridine nucleotide which is at its active center. This prosthetic group can be removed from the enzyme only by treatment which causes denaturation of the protein. Oxidation and reduction of the pyridine nucleotide can be measured by changes in absorbance at 345 rnb and by fluorescence. The catalytic activity is believed to be a direct transfer of reducing equivalents from the a-hydroxy acid to the keto acid mediated by the pyridine nucleotide. The oxidized enzyme in the absence of substrate undergoes a spontaneous reduction as measured by an increased fluorescence of the prosthetic group which is probably catalyzed by some groups on the enzyme itself. The noninvolvement of this spontaneous reduction in the catalytic transhydrogenation reaction is discussed.