Feldman R I, Boyer P D
J Biol Chem. 1985 Oct 25;260(24):13088-94.
Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.
已知分离出的叶绿体偶联因子1 ATP酶每摩尔酶保留约1摩尔紧密结合的ADP。一些实验结果表明结合的ADP位于催化位点，但当添加CaATP或MgATP时结合的ADP缓慢置换的观察结果并不支持这一观点。本文报道的实验表明了在催化位点结合的ADP为何会出现缓慢置换。当用紧密结合的[3H]ADP标记的偶联因子1在添加MgATP或CaATP之前暴露于Mg2+或Ca2+时，会观察到ATP水解开始时出现明显延迟，并且[3H]ADP仅缓慢置换。Mg2+或Ca2+能非常迅速地诱导抑制作用，就好像每当带有紧密结合ADP的酶在ATP之前遇到Mg2+或Ca2+时就会产生一种受抑制的酶形式。通过与EDTA温育，受抑制的形式可被缓慢重新激活，尽管会遇到一些不可逆的活性损失。相反，当将MgATP或CaATP添加到通过与EDTA温育而耗尽Mg2+和Ca2+的酶中时，ATP水解迅速开始，并且大部分紧密结合的[3H]ADP在几秒钟内释放，这正如在催化位点结合时所预期的那样。在有利于向含有紧密结合ADP的位点添加Pi的条件下与Pi温育，可在很大程度上防止Mg2+诱导的ATP酶活性抑制以及紧密结合的[3H]ADP不被置换。如果当没有伴随Pi的MgADP或CaADP以高亲和力构象紧密结合在酶的三个催化位点之一上时酶催化受到极大阻碍，那么我们的结果和其他结果就能得到解释。
