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J. Biol. Chem., Vol. 283, Issue 33, 22541-22549, August 15, 2008

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

Yuta Hatori, Ayami Hirata, Chikashi Toyoshima, David Lewis, Rajendra Pilankatta, and Giuseppe Inesi¹

From the Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan and the California Pacific Medical Center Research Institute, San Francisco, California 94107

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 [-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 (NMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [-³²P]ATP by the NMBD 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 NMBD mutant, evidently due to Cu⁺ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in NMBD 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.

Received for publication, April 8, 2008 , and in revised form, June 5, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant NHLBI RO301–69830 (to G. I.). This work was also supported by a Special Promoted Project grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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¹ To whom correspondence should be addressed: 475 Brannan St., San Francisco, CA 94107. Tel.: 415-600-1745; Fax: 415-600-1725; E-mail: ginesi{at}cpmcri.com.

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