Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum

‘Malic’ enzyme (l-malate:NADP⁺ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) was purified from Clostridium thermocellum by DEAE-cellulose, agarose-NADP and Sephadex G-200 column chromatography. The 117-fold purified ‘malic’ enzyme displayed a maximum activity of 135 units/mg at 40°C and represented 0.8% of the total cell protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of the protein suggested 90% purity and an approximate tetrameric subunit molecular weight of 40 000. The enzyme absolutely required both bivalent and monovalent cations for catalysis. Mn²⁺ and NH₄⁺ were the most effective cationic activators examined. Increasing NH₄⁺ concentration increased both enzyme activity and affinity toward l-malate. The apparent K_m for l-malate was 3 · 10⁻⁴ M at 0.4 mM NH₄Cl. Enzyme activity increased linearly when temperature was raised between 22–60°C and a Q₁₀ of 2.1 was calculated from an Arrhenius plot. The enzyme was stable to heating at 60°C but was denatured at higher temperatures. The enzyme half-life was 10 min at 72°C. The enzyme displayed a broad pH optimum (7.2–8.2 for Tris-HCl buffer) but was inactivated by p-chloromercuribenzoate. The high thermal stability, low apparent molecular weight and NH₄⁺ activation are properties not common to all previously described ‘malic’ enzymes.