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 degrees 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 Km for L-malate was 3 X 10(-4) M at 0.4 mM NH4Cl. Enzyme activity increased linearly when temperature was raised between 22-60 degrees C and a Q10 of 2.1 was calculated from an Arrhenius plot. The enzyme was stable at heating at 60 degrees C but was denatured at higher temperatures. The enzyme half-life was 10 min at 72 degrees C. The enzyme displayed a broad pH optimum (7.2-87.2 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.