Abstract
We report in this paper that highly purified Escherichia coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase are inactivated by O2- with second order rate constants in the range of 10(6) to 10(7) M-1 s-1. Each of these enzymes belongs to the hydro-lyase class and contains catalytically active [4Fe-4S] clusters. Simultaneous with inactivation by O2- is the release of iron from their clusters. Our working hypothesis is O2- inactivates these enzymes by oxidizing their clusters to an unstable oxidation state, and cluster degradation follows. Consistent with this hypothesis is our observation that spinach dihydroxy-acid dehydratase, a member of the hydro-lyase class that has a catalytically active [2Fe-2S] cluster, is not inactivated and does not lose iron in the presence of O2-. Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is also not inactivated by O2-. We also report the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1. E. coli dihydroxy-acid dehydratase has been reported previously to be inactivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions. We report here that E. coli fumarase A, fumarase B, and aconitase are also inactivated in vivo by hyperbaric O2. Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated.
Highlights
Branched-chain amino acand the rate constants for the inactivation of E. coli ids are antidotes because hyperbaric O2and paraquat lead to dihydroxy-acid dehydratase and mammalian aconitase the inactivation of the third enzyme in the branched-chain by Hz02are about lo3M - ~s-l
E. coli dihydroxy-acidde- amino acid biosynthetic pathway, dihydroxy-acid dehydratase hydratase has been reported previously to be inacti- (Brown and Yein, 1978; Fee et al, 1980). vated in vivo when cells are grown in hyperbaric 02, Dihydroxy-acid dehydratase is a member of the hydro-lyase presumably due to the increased 0; generated under class of enzymes and catalyzes the dehydratiroenaction shown these conditions
E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, mammalian aconitase, spinach dihydroxy-aciddehydratase, and porcine fumarase were individually added to the 0;generating mixtures soon after the addition of xanthine oxidase.Subsamples of the mixtures were withdrawn periodieally and immediately assayed for the hydro-lyase activity remaining by diluting the subsample (>30x) into substratecontaining assay mixture which served to stop the inactivation
Summary
Materials-All purchased chemicals were the best grade available. The substrate 2,3-dihydroxy-3-methylbutyricacid was made by cis-hydroxylation of 3-methyl-2-butenoicacid with potassium permanganate (Nielsenet al., 1969)which givesa racemic mixture. E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, mammalian aconitase, spinach dihydroxy-aciddehydratase, and porcine fumarase were individually added to the 0;generating mixtures soon after the addition of xanthine oxidase.Subsamples of the mixtures were withdrawn periodieally and immediately assayed for the hydro-lyase activity remaining by diluting the subsample (>30x) into substratecontaining assay mixture which served to stop the inactivation. ( dihydroxy-aciddehydratase activity in the strainwith the pRL50 plasmid is higher, the fraction of the dihydroxy-aciddehydratase activity that is inactivated by hyperbaric 0, is very similar in both strains.) For fumarase A, E. coli strain JRG1905 was used, and, for fumarase B, JRG 2034 was used These strains were kindly supplied by John Guest of the University of Sheffield and are transformants of E. coli JH400, a fimAC fumB deletion strain, containing in the case of JRG 1905 the fumA plasmid, pGS57, and, in the case of JRG 2034, the fume plasmid, pGS93 (Woods et al, 1988). The colored complex between Fez+ anfderene was measured by determining the absorbance at 562 nm and comparing it to a standard curve
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