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首页> 外文期刊>Biochemistry >Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH
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Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

机译：高pH下氮酶MoFe蛋白的周转依赖性失活

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Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725?13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanismbased transformation that may provide a new probe of nitrogenase catalysis.
机译：质子的吸收伴随着所有已知底物被固氮酶的还原。因此，较高的pH值将限制质子作为周转必不可少的底物的可用性，从而增加酶的高度还原形式的比例，以供进一步研究。鉴于Pham和Burgess [（1993）Biochemistry 32，13725？13731]报道的观察结果表明，MoFe蛋白在pH 8.65以上会发生不可逆的蛋白质变性，因此高pH方法的实用性似乎是有问题的。相反，我们通过酶活性和晶体学分析发现，当在pH 9.5下孵育时，MoFe蛋白是稳定的。但是，我们确实观察到，在较高的pH值和周转条件下，MoFe蛋白会缓慢失活。尽管在这些条件下会发生正常的底物还原水平，尽管水平很低，但MoFe蛋白却经历了复杂的转化。最初，该酶在pH 9.5时可逆地抑制底物还原，但是在第二个较慢的过程中，MoFe蛋白不可逆转地失活，这是通过在最佳pH值为7.8时的底物还原活性来测量的。与天然MoFe蛋白质相比，最终灭活的MoFe蛋白质具有更大的流体力学半径，但它具有铁和钼的完整补体。重要的是，通过ATP水解的支持以及在ATP和铝存在下与Fe蛋白形成紧密的复合物，判断出修饰的MoFe蛋白保留了与其固氮酶伴侣Fe蛋白特异性相互作用的能力。氟化物。依赖于营业额的失活与构象变化相结合，表明了一种基于机理的转化，可提供一种新的固氮酶催化探针。

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