Physical and chemical properties of reversibly inactivated lactate dehydrogenases.

The effect of reversible inactivation on the physical and chemical properties of dogfish M4 and chicken heart H* lactate dehydrogenases has been studied. Reduced diphosphopyridine nucleotide was found to have a significant effect on the rate of recovery of the enzymatic activity, but not on the over-all recovery. Optical rotatory dispersion, circular dichroism, fluorescence, and immunological studies have shown structural differences between the native and reassociated enzyme. Further evidence for the difference in conformational structure was obtained from the pattern of reactivity of sulfhydryl groups of the reactivated enzyme. The native enzyme was also found to be more stable to heat and urea denaturation than the reassociated form. Pyruvate inhibition, and the catalytic activity with coenzyme analogues, was found to be different for the native and the reassociated enzyme. These results indicate the possibility that a conformational difference exists between the native and the reassociated enzyme for both dogfish M4 and chicken Hh lactate dehydrogenase. Studies on the lactate dehydrogenase reassociated in the presence of reduced DPN showed structural characteristics which are closer to the native dehydrogenase than the enzyme which was reassociated in the absence of the reduced coenzyme. Incubation of the enzyme with DPNH after reassociation does not convert the reassociated enzyme to a structure similar to that of the native enzyme. These studies suggest a possible role of the reduced coenzyme in the folding of the peptide chain or in the subunit interaction and in the attainment of the native conformation of the enzyme.