ATP Hydrolysis and Phosphoenzyme Turnover of the Spf1 P5-ATPase

ADAMO HUGO PEDRO; CORRADI GERARDO RAUL; MAZZITELLI LUCIANA ROMINA; DE TEZANOS PINTO FELICITAS

Snowmass Village, Colorado

Conferencia; New Frontiers in transport ATPases: From Mechanistic to Therapeutic concepts; 2012

FASEB-Federation of American Societies for Experimental Biology

ATP Hydrolysis and Phosphoenzyme Turnover of the Spf1 P5-ATPase.  Hugo P. Adamo, Gerardo R. Corradi, Luciana R. Mazzitelli and Felicitas de Tezanos Pinto. From the Instituto de Química y Fisicoquímica Biológicas (IQUIFIB).  Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.    The P5-ATPases have been shown to influence protein biogenesis, folding and transport and have been linked to human diseases. Still they are the least characterized at the biochemical level. We overexpressed in Saccharomyces cerevisiae and purified the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end.  Like the Spf1, GFP-Spf1 was localized in the yeast endoplasmic reticulum. The purified GFP-Spf1 protein was able to hydrolyze ATP at a rate of about 0.3-1 mmol Pi/mg/min in a simple reaction media containing no added metal ions except Mg2+.  The Mg2+ dependency of the GFP-Spf1 ATPase was similar to that of other P-ATPases were Mg2+ acts as a cofactor. No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. The ATPase activity of GFP-Spf1 was similar in both phosphatidylcholine or brain acidic phospholipids, and it decreased when Mn2+ was added to the reaction media.  In the presence of MgATP, GFP-Spf1 formed a stable phosphoenzyme intermediate (EP). The amount of phosphoenzyme did not change in the presence of   Ca2+ or a mixture or acidic lipids.  When chased by the addition of cold ATP 90 % of EP remained stable after 5 s suggesting that the forward processing of the phosphoenzyme was impaired. In contrast, less than 20% of EP was present 3 s after the addition of ADP. The ability of ADP greatly accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme.  This behavior of Spf1 may reflect that a proper countertransported substrate was limiting or the need for a regulatory “specificity” factor.