Inhibition of the Formation of the Spf1p Phosphoenzyme by Ca2+

CORRADI, G.R.; SARBIA N; CZYSEZON NA; ADAMO HP; MAZZITELLI LR

JOURNAL OF BIOLOGICAL CHEMISTRY (ONLINE)

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2016 vol. 291 p. 7767 - 7773

1083-351X

P5-ATPases are important for processes associated with theendosomal-lysosomal system of eukaryotic cells. In humans, theloss of function of P5-ATPases causes neurodegeneration. Inthe yeast Saccharomyces cerevisiae, deletion of P5-ATPaseSpf1p gives rise to endoplasmic reticulum stress. The reactioncycle of P5-ATPases is poorly characterized. Here, we showedthat the formation of the Spf1p catalytic phosphoenzyme wasfast in a reaction medium containing ATP, Mg2, and EGTA.Low concentrations of Ca2 in the phosphorylation mediumdecreased the rate of phosphorylation and the maximal level ofphosphoenzyme. Neither Mn2 nor Mg2 had an inhibitoryeffect on the formation of the phosphoenzyme similar to that ofCa2. The Km for ATP in the phosphorylation reaction was 1M and did not significantly change in the presence of Ca2. Halfmaximalphosphorylation was attained at 8 M Mg2, but higherconcentrations partially protected from Ca2 inhibition. In conditionssimilar to those used for phosphorylation, Ca2 had a smalleffect accelerating dephosphorylation and minimally affectedATPase activity, suggesting that the formation of the phosphoenzymewas not the limiting step of the ATP hydrolytic cycle.