Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima
Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima
复制标题
DOI：
10.1074/jbc.m802735200
复制
发表时间：
2008-08-15
影响因子：
4.8
通讯作者：
Inesi, Giuseppe
中科院分区：
文献类型：
--
作者：
Hatori, Yuta;Hirata, Ayami;Inesi, Giuseppe
重组和纯化的Thermotoga maritima CopA在Cu+ （pH 6, 60℃）存在时atp酶速度为1.78 ~ 2.73 mu mol/mg/min，在Cu+不存在时atp酶速度为0.03 ~ 0.08 mu mol/mg/min。在没有Cu+的情况下，利用[γ - p -32]ATP可以获得高水平的酶磷酸化。这种磷酸酶的衰变速度比铜慢得多。事实上，在加入Cu+后，由于快速水解裂解，磷酸化酶被还原到更低的稳态水平。CopA在其n -金属结合域（Delta NMBD）缺失或NMBD半胱氨酸（CXXC）突变后，可忽略不计的atp酶周转可由CopA维持。然而，Delta NMBD和CXXC突变体通过利用[γ - p -32]ATP获得了高水平的磷酸酶，而Cu+对其形成或水解裂解没有影响。利用Pi也可以获得磷酸酶形成（E2P），即使在Delta NMBD突变体中，该反应也被Cu+ （E2到E1的转变）所抑制，这显然是由于Cu+结合在NMBD以外的（运输）位点上。E2P在Delta NMBD中水解裂解更快，而在CXXC突变体中较慢。我们认为Cu+与NMBD结合是产生CopA“活性”构象所必需的，因此额外的Cu+结合到另一个（跨膜运输）位点启动更快的周期，包括Cu的形成。与P相似的E1，其次是与P相似的E1到E2-P的构象转变和磷酸盐的水解解理。H479Q突变（类似于Wilson病中发现的突变）使CopA无法利用ATP，而P-i的磷酸化被保留。
Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 mu mol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 mu mol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-P-32]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 similar to 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 (Delta NMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-P-32]ATP by the Delta 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 Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the Delta NMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in Delta 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 similar to P, followed by the E1 similar to 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.