P-nitrophenylphosphatase activity of plasma membrane H(+)-ATPase from yeast. Implications for the regulation of the catalytic cycle by H+.

Abstract

The H(+)-ATPase from Schizosaccharomyces pombe belongs to the group of transport ATPases which displays two main conformational states, E1 and E2 (P-type ATPase). In this report, we show that, as in the case of other P-type ATPase, the purified enzyme exhibits a p-nitrophenylphosphatase activity which can be completely inhibited by vanadate. In aqueous medium, p-nitrophenyl phosphate hydrolysis proceeds at only 0.5% of the rate of ATP hydrolysis, and both activities can be stimulated 3- to 4-fold by decreasing the pH from 7.5 to 6.5. Addition of the organic solvent dimethyl sulfoxide (10-40%), which has been shown to favor the E2 conformation, stimulates the p-nitrophenylphosphatase activity but inhibits the ATPase activity. At pH 7.5, the Km for p-nitrophenyl phosphate decreases when dimethyl sulfoxide is present. In the presence of 30% (v/v) dimethyl sulfoxide, the phosphatase activity can be inhibited by ATP (K(i) 300 microM) or by P(i) (K(i) 1 mM). The H(+)-ATPase incorporated into liposomes retains pNPPase activity, but it does not support H+ transport. Gel electrophoresis reveals that the pattern of H(+)-ATPase cleavage by trypsin changes when vanadate, Me2SO, or both compounds are present in the medium, regardless of the pH used during trypsinization. We propose that p-nitrophenyl phosphate is hydrolyzed by a H(+)-ATPase conformation distinct from that which hydrolyzes ATP, most probably an E2-like form. We also suggest that, in addition to the E1-E2 transition, the enzyme activity can be regulated by protons at another step of the catalytic cycle.