Purification and properties of malate dehydrogenase (Decarboxylating) from mycobacterium 607

Abstract

Malate dehydrogenase (decarboxylating) (l-malate: NADP oxidoreductase (decarboxylating), EC 1.1.1.40) has been purified 100-fold from Mycobacterium 607, and its properties for oxidative decarboxylation reaction have been studied. The pH optimum was 7.8. Below pH 5.5 the enzyme was unstable. Reaction with NAD was slow. K₃ for NADP was 4·10⁻⁵ M.

Bivalent cations such as Mn²⁺, Mg²⁺, Co²⁺ or Ni²⁺ were essential for activity. The order and extent of effectiveness of these cations depended on the concentration of malate and Ks⁺. K⁺ activated the reaction, but higher substrate concentrations reduced or inhibitory. This inhibition was reversed completely by GSH and partially by increasing the activity bivalent cation concentration. The involvement of a sulfhydryl group in the enzymic reactions is also suggested by other inhibition studies.

The apparent K_s for malate at pH 7.4 was 1☆10⁻³ M and at 8.2 it was 2.5·10⁻³ M. Higher malate concentrations were inhibitory. Raising the pH lowered, and increasing the Mg²⁺ concentration abolished, this effect. The cause of substrate inhibition is concluded to be its chelatioon of bivalent cation.

Anions also affected the activity. Activity with Cl- was more than that with SO₄²⁻ and this effect was more marled at higher pH.