PubMed2001
In the amino-acid-fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A-bound carboxymethyl-selenoether. We investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation. After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep-tag proteins. GrdD was the only subunit that catalysed arsenate-dependent hydrolysis of acetyl phosphate (up to 274 U.mg-1), whereas GrdC was completely inactive. GrdD contained two cysteine residues that were exchanged by site-directed mutagenesis. The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive. Next, these thiols were analysed further by chemical modification. After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected. Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated. Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry. Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein. This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate.
Amino Acid Oxidoreductases↗