Intermediate Phosphorylation Reactions in the Mechanism of ATP Utilization by the Copper ATPase (CopA) of Thermotoga maritima

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-(32)P]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-(32)P]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 P(i), 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 P(i) is retained.