Whittaker Mei M, Whittaker James W
Department of Science and Engineering, School of Medicine, Oregon Health and Science University, 20000 Northwest Walker Road, Beaverton, Oregon 97006-8921, USA.
Biochemistry. 2008 Nov 4;47(44):11625-36. doi: 10.1021/bi8015636. Epub 2008 Oct 9.
Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37 degrees C) shows it is linked to ionization of a single proton with a p K a of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20 degrees C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.
体外无锰超氧化物歧化酶对金属的摄取是一个复杂的过程，呈现出多相“门控”反应动力学以及显著的S形温度曲线，这导致了一种构象门控金属结合模型，该模型要求在“封闭”和“开放”形式之间进行转换。这项工作系统地探索了野生型（WT）脱辅基蛋白及其突变变体中金属结合的结构决定因素，以此作为对机理模型的检验。在生理条件（37℃）下金属化的pH依赖性表明，它与单个质子的电离有关，其pKa为7.7。尺寸排阻色谱表明，即使脱辅基蛋白完全转化为开放形式，它也是二聚体。通过使用二硫键工程将共价限制引入蛋白质中，探究了分子运动在金属结合中的作用。虽然限制结构域界面处的运动没有影响，但限制亚基界面会显著干扰金属摄取，但不会阻止这一过程。活性位点环境中残基的诱变导致转变温度大幅变化多达20℃或pH敏感性丧失。基于这些结果，提出了一种锰超氧化物歧化酶摄取金属的机制，该机制涉及活性位点残基重新定向以形成金属进入通道。
