Tricarboxylic acid cycle and related enzymes in cell-free extracts of Mycobacterium tuberculosis H37Rv

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Abstract
Washed whole-cell suspensions of Mycobacterium tuberculosis H37Rv oxidized only pyruvate, acetate, oxaloacetate and lactate to a significant extent. Sonic extracts of the organism utilized lactate and oxaloacetate in the presence of a suitable artificial electron acceptor (phenazine, methosulphate or menadione) and citrate, cis-aconitat and isocitrate only after the addition of NADP+ (di-phosphoadenine nucleotide) as well as the electron acceptor. All enzymes of the tricarboxylic acid cycle and of the electron-transport chain (with the exception of cytochrome oxidase) were demonstrated to be present in the crude cell-free extracts. Enzymes of the gly-oxylate bypass were present in the cell-free extracts. Isocitratase was present in extracts of the glycerol-grown organisms. All the other dehydrogenases except for malate dehydrogenase were found to be NADP+- but not NAD+-dependent. Malate dehydrogenase was dependent on NAD+ only at high pH values where NADH-oxidase activity is very low. The oxidation of NADH very rapid compared with that of NADPH was insensitive to cyanide (presumably because cytochrome oxidase is absent). Vitamin K3 but not vitamin K1 stimulated the oxidation of NADH. NADH-cytochrome c-reductase activity lost on dialysis was restored by vitamin K or FMN (flavin mononucleotide) but not by vitamin K or FAD (flavin adenine di-nucleotide). NADPH-cytochrome c-reductase activity decreased after dialysis was restored by vitamin K3 alone but not by vitamin K1, FAD or FMN.