Inhibition of the formation of the spf1p phosphoenzyme by Ca2+

Abstract

P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeast Saccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg2+, and EGTA. Low concentrations of Ca2+ in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn2+ nor Mg2+ had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca2+. The Km for ATP in the phosphorylation reaction was 1 μM and did not significantly change in the presence of Ca2+. Halfmaximal phosphorylation was attained at 8 μM Mgμ, but higher concentrations partially protected from Caμ inhibition. In conditions similar to those used for phosphorylation, Ca2+ had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.