The Parkinsons associated P5-ATPase Spf1 is inactivated by Zn2+

GUIDO D. PETROVICH; GERARDO R. CORRADI ; ADAMO HUGO P.

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

P-type ATPases (P1 to P5) are integral membrane proteins that are responsible for the active transport of different ligands. P5-ATPases are the least studied members of the whole family of P-ATPases and its transported substrate remains unknown. In humans, mutations in the P5-ATPases genes are implicated in neurological disorders, as the Kufor-Rakeb syndrome, a Parkinsonism with dementia, hereditary spastic paraplegia, neuronal ceroid lipofuscinosis, autism and intellectual disability. While the mechanism behind these pathologies is not well understood recent studies suggest a complex interaction between genetic predisposition and environmental factors. In this line, several Parkinson?s associated genes have been shown to cause loss of metal ion homeostasis. We have measured the effect of metals on the ATPase activity of the micellar preparation of purified recombinant Spf1 P5A-ATPase from the simple eukaryote Saccharomyces cerevisiae. The addition to the reaction media of Ca2+, Mn2+ and Co2+ reversible inhibited the enzyme with Ki >100 M. In contrast, preincubation of the protein with low concentrations of Cd 2+ or Zn2+ led to the loss of ATPase activity that could not be reverted by a subsequent treatment with EGTA or DTT. The inactivation by Zn2+ was not altered by Mg2+. In contrast, catalytic concentrations of ATP during preincubation protected from Zn2+ inactivation. Similar protection was attained by adding ADP instead of ATP, suggesting that protection was due to the binding of the nucleotide to Spf1. Kinetic analysis indicates that the the modulation of the catalytic efficiency by high concentrations of ATP was the most affected parameter in the Zn2+-treated enzyme. These results are consistent with the hypothesis that the modification of a target residue at the catalytic nucleotide binding domain of the enzyme prevents the acceleration by ATP of the conformational transition of the enzyme from E2 to E1. With grants from UBA, CONICET and ANPCyT