Activation of acetate in acetate-grown Methanosarcina thermophila: purificationm and characterization of acetate kinase
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Extracts of acetate-grown Methanosarcina thermoghila were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 Î¼moles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 Î¼moles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an Î±2 homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70Â°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 Î¼moles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+.
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