Intermediate Phosphorylation Reactions in the Mechanism of ATP Utilization by the Copper ATPase (CopA) of Thermotoga maritima
| Autor: | Giuseppe Inesi, Yuta Hatori, Chikashi Toyoshima, Rajendra Pilankatta, Ayami Hirata, David Lewis |
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| Rok vydání: | 2008 |
| Předmět: |
Stereochemistry
ATPase Cleavage (embryo) Biochemistry Phosphates Substrate Specificity Bacterial Proteins Lecithins Thermotoga maritima Phosphorylation Cation Transport Proteins Molecular Biology Adenosine Triphosphatases chemistry.chemical_classification biology Chemistry Cell Biology Membrane transport Phosphoproteins biology.organism_classification Kinetics Membrane Transport Structure Function and Biogenesis Crystallography Enzyme Copper-Transporting ATPases biology.protein Steady state (chemistry) Copper Binding domain |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m802735200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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