Dataset Information

Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex.

ABSTRACT: Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.

SUBMITTER: Williams KP

PROVIDER: S-EPMC1131392 | biostudies-other | 1990 May

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5'-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.

Dataset Information

Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets