Analysis of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE.
The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K(m) being 25 micro m and the k(cat) 1.7 x 10(-3) s(-1). Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K(m) (47 micro m) but not significantly the k(cat) (2.1 x 10(-3) s(-1)). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.
Blokesch
Melanie
M
Department Biologie I, Mikrobiologie, Ludwig-Maximilians-Universität München, München, Germany.
Paschos
Athanasios
A
Bauer
Anette
A
Reissmann
Stefanie
S
Drapal
Nikola
N
Böck
August
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D014867:Water / Q000737:chemistry*
D001426:Bacterial Proteins; C108878:HypF protein, Bacteria; C085871:hypE protein, Bacteria; D014867:Water; D000255:Adenosine Triphosphate; D006864:Hydrogenase; D000251:Adenosine Triphosphatases