Al-Shawi M K, Ketchum C J, Nakamoto R K
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22906-0011, USA.
Biochemistry. 1997 Oct 21;36(42):12961-9. doi: 10.1021/bi971478r.
The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.
大肠杆菌F(\text{O})F(1) ATP合酶的解偶联突变体gammaM23K被发现可增加gamma亚基与beta亚基之间的相互作用能，阻止结合能的正确利用以驱动催化作用，并使该酶处于磷酸根释放模式。本文评估了此突变对协同ATP合成中底物结合的影响。测定了gammaM23K F(\text{O})F(1)由ADP和磷酸根激活ATP合成的情况。ADP的半饱和常数（K({0.5})）未受影响，但磷酸根的K({0.5})约高7倍，尽管表观最大反应速度（V({max})）接近野生型水平。野生型酶在pH 7.5和30℃时的转换数为82 s(^{-1})。在氧化磷酸化过程中，ATP的表观解离常数（K(\text{I})）未受影响，野生型和gammaM23K酶的K(\text{I})均为5 - 6 mM。因此，在存在质子动力势（ΔμH(^+)）的情况下，ATP的表观结合亲和力比在高亲和力催化位点测得的亲和力降低了7个数量级。对gammaM23K F(\text{O})F(_1)的ATP合成进行阿累尼乌斯分析表明，与ATP水解一样，过渡态的焓变（ΔH）更正，熵变（TDeltaS）更负，两者相加吉布斯自由能变化（ΔG）变化不大。这些结果表明，ATP合成效率低下是因为gamma亚基与beta亚基之间存在额外的键，必须打破此键才能达到过渡态。使用等动力学曲线分析过渡态结构表明，ATP水解和合成利用相同的动力学途径。将此信息纳入旋转催化模型表明，在底物浓度饱和时，水解和合成的限速步骤是旋转动力冲程，此时每个beta亚基都会改变构象和对核苷酸的亲和力。
