Beef Heart Malic Dehydrogenases

V. A KINETIC STUDY OF THE REACTION CATALYZED BY THE SUPERNATANT ENZYME

From the ¹ From the Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, New York 10461

Kinetic studies on beef heart supernatant malic dehydrogenase were carried out in 0.1 m triethanolamine-acetate buffer. Analysis of the data by the method of Dalziel revealed that specific relationships between calculated kinetic parameters did not conform to the criteria proposed for mechanisms involving sequential addition for each pair of substrates. The results also did not conform to the criteria proposed for a mechanism involving a random order of substrate addition to enzyme, in which all of the complexes are in rapid equilibrium with the substrates. These observations can be contrasted with those of Raval and Wolfe (1) on hog heart malic dehydrogenase. With this enzyme, of apparent mitochondrial origin, the results were found to be compatible with a mechanism involving an ordered addition of substrate to the enzyme, with no kinetically active ternary complexes.

A kinetic mechanism for supernatant malic dehydrogenase has been proposed; it involves the participation of three binary complexes of substrate and enzyme (enzyme-oxaloacetate, enzyme-diphosphopyridine nucleotide, and enzyme-DPNH) and no kinetically significant ternary complexes. It has further been proposed that the preferred route of the reaction is via the branch of the pathway involving enzyme-DPNH, and that the rate constants are identical for DPNH interacting either with free enzyme or with the enzyme-oxaloacetate complex. This formulation is consistent with the experimentally determined kinetic parameters and with the independently determined dissociation constant for enzyme-DPNH obtained by fluorometric measurements.

Substrate inhibition of supernatant malic dehydrogenase by oxaloacetate was observed at pH values below 7.8. This observation may be interpreted as further indication of the existence of a binary enzyme-oxaloacetate complex. However, the possibility that the inhibition is due to the formation of inactive ternary complexes with oxaloacetate cannot be ruled out.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?