Taiz L, Nelson H, Maggert K, Morgan L, Yatabe B, Taiz S L, Rubinstein B, Nelson N
Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Biochim Biophys Acta. 1994 Sep 14;1194(2):329-34. doi: 10.1016/0005-2736(94)90315-8.
The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.
酵母液泡ATP酶的A亚基包含三个高度保守的半胱氨酸：Cys-261、Cys-284和Cys-538。Cys-261位于核苷酸结合P环内。通过定点诱变将每个保守的半胱氨酸以及一个非保守的半胱氨酸Cys-254都改变为丝氨酸，并测定了其对pH 7.5条件下生长的影响。Cys-254→Ser、Cys-261→Ser以及双突变体在pH 7.5条件下均能生长，并且具有对硝酸盐和巴弗洛霉素敏感的ATP酶活性。然而，Cys-261→Ser和双突变体的ATP酶活性对巯基抑制剂N-乙基马来酰亚胺不敏感，这表明Cys-261是N-乙基马来酰亚胺的抑制位点。将Cys-284或Cys-538改变为丝氨酸会阻止在pH 7.5条件下的生长。因此，Cys-284和Cys-538似乎是组装或催化所必需的关键半胱氨酸残基。
