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 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 micromol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 approximately 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 (DeltaNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-3)P]ATP by the DeltaNMBD 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 DeltaNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in DeltaNMBD 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 approximately P, followed by the E1 approximately 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.
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Yuta
Y
Hatori
Ayami
A
Hirata
Chikashi
C
Toyoshima
David
D
Lewis
Rajendra
R
Pilankatta
Giuseppe
G
Inesi
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D007700:Kinetics; D054709:Lecithins / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D054709:Lecithins; D010710:Phosphates; D010750:Phosphoproteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases