Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima

Abstract

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 μmol/mg/min in the presence of Cu+ (pH 6, 60 °C) and 0.03-0.08 μmol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [γ- 32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu·E1 ∼ P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (ΔNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [γ-32P]ATP by the ΔNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the ΔNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in ΔNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu·E1 ∼ P, followed by the E1 ∼ P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.