Zhou J M, Boyer P D
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
J Biol Chem. 1993 Jan 25;268(3):1531-8.
Previous results have not established whether the attainment of a rapid photophosphorylation rate as ADP concentration is increased in the micromolar range (apparent Km = approximately 30 microM) results from the filling of a second or a third catalytic site. Measurements reported here show that the ATP synthase of chloroplast thylakoids, with 2-4 microM medium ADP present during steady-state photophosphorylation, has one catalytic site filled with tightly bound nucleotides, but other catalytic sites are largely empty. Thus, the rapid increase in the photophosphorylation rate with higher ADP concentrations results from the filling of a second catalytic site. Even with 30 microM added ADP in the dark, the binding of more than one ADP per synthase was not detectable. The sensitivity of the assay was such that the Kd for binding of ADP at a second catalytic site of the de-energized synthase is > 150 microM, considerably above the apparent Km for rapid photophosphorylation. This result can be explained by an increase in the affinity of a second catalytic site for ADP upon energization. Other experiments assessed the effect of ADP binding at a second catalytic site on the equilibrium between bound ATP and ADP and P(i) at the tight catalytic site. When the rate of photophosphorylation is limited by a low ADP concentration, about equal amounts of ATP and ADP are bound at one catalytic site on the synthase. In contrast, when the rate is limited by a low P(i) concentration with 100 microM ADP present, the equilibrium of bound reactants is shifted so that close to one ATP per synthase is present. This is as expected if the binding of ADP at a second catalytic site allows the protonmotive force to promote ATP formation from ADP and P(i) at a tight binding catalytic site. A scheme for the binding change mechanism incorporating these results is presented.
先前的研究结果尚未确定，当在微摩尔范围内增加ADP浓度（表观Km = 约30 μM）时，快速光磷酸化速率的实现是由于第二个还是第三个催化位点被占据。本文报道的测量结果表明，在稳态光磷酸化过程中，叶绿体类囊体的ATP合酶在存在2 - 4 μM中等浓度ADP的情况下，有一个催化位点被紧密结合的核苷酸占据，但其他催化位点基本为空。因此，随着ADP浓度升高，光磷酸化速率的快速增加是由于第二个催化位点被占据。即使在黑暗中加入30 μM ADP，也检测不到每个合酶结合不止一个ADP。该检测方法的灵敏度表明，去能合酶第二个催化位点结合ADP的解离常数（Kd）> 150 μM，远高于快速光磷酸化的表观Km。这一结果可以通过第二个催化位点在被激活后对ADP亲和力增加来解释。其他实验评估了第二个催化位点上ADP结合对紧密结合催化位点上ATP与ADP和无机磷酸（Pi）之间平衡的影响。当光磷酸化速率受低ADP浓度限制时，合酶的一个催化位点上结合的ATP和ADP量大致相等。相反，当速率受低Pi浓度限制且存在100 μM ADP时，结合反应物的平衡发生移动，使得每个合酶接近一个ATP。如果第二个催化位点上ADP的结合能使质子动力促进ADP和Pi在紧密结合催化位点上形成ATP，那么这是预期的结果。本文提出了一个包含这些结果的结合变化机制方案。
