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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase.

The kinetics of H-D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D2 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H2, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8. 5. The rapid formation of H2 with respect to HD in the D2/H2O system is consistent with a heterolytic cleavage of D2 into D+ and an enzyme hydride that can both exchange with the solvent. The H-D-exchange activity was lower in the H2/D2O system than in the D2/H2O system. The other reactions catalyzed by the hydrogenase, H2 oxidation and H2 evolution, are pH dependent; the optimal pH were 9.5 for H2 uptake and 4.0 for H2 production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H-D exchange. When iodo[1-14C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H2 than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H2-activated hydrogenase from T. roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.[1]

References

  1. Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase. Zorin, N.A., Dimon, B., Gagnon, J., Gaillard, J., Carrier, P., Vignais, P.M. Eur. J. Biochem. (1996) [Pubmed]
 
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