The kinetic properties of nad-linked malic enzyme from muscle of the dung beetle Catharsius

Abstract

Nad-linked malic enzyme (L-malate: NAD+ oxidoreductase (decarboxylating), E.C. 1.1.1.38) was purified from muscle of the dung beetle Catharsius to a specific activity of 28 μmol/min/mg of protein. Its molecular weight was estimated as 250,000–260,000 by Sephadex gel filtration. Activity was dependent on Mg2+ or Mn2+, for which Full-size image (<1 K) values of 0.18 and 0.019 mM were obtained respectively. With Mg2+, at pH 7.2, apparent Full-size image (<1 K) values obtained for L-malate (with 0.76 mM NAD+) and NAD+ (with 2.5 mM L-malate) were 1.40 and 0.54 mM respectively. Plots of activity versus substrate concentration tended to be sigmoidal, but became hyperbolic in the presence of succinate or fumarate. These dicarboxylic acids lowered apparent Full-size image (<1 K) values, but did not affect maximum velocity. With succinate present, the apparent Full-size image (<1 K) for NAD+ decreased with increasing L-malate concentration towards a limiting value of 0.12 mM; the apparent Full-size image (<1 K) for L-malate decreased with increasing NAD+ concentration towards a limiting value of zero. The maximum velocity decreased sharply below pH 7.0; apparent Full-size image (<1 K) values increased with increasing pH. With Mn2+, plots of activity versus substrate concentration were more nearly hyperbolic and apparent Full-size image (<1 K) values were lower than with Mg2+. With NADP, maximum activities 30 and 40% those with NAD were obtained with Mg2+ and Mn2+ respectively. The reductive carboxylation of pyruvate occurred in the presence of Mn2+, and with high concentrations of bicarbonate and pyruvate, at up to 5% the maximum rate of the forward reaction. A Ca2+ stimulated oxaloacetate decarboxylase activity was associated with the enzyme.