Novel Substrate Oxidation by Enzyme Engineering

描述（由申请人提供）：了解P450所显示的多种化学性质是生物医学科学的一个核心问题，需要了解它们臭名昭著的特异性和混杂性倾向。药物代谢P450在底物识别中显示出显着的混杂性，而参与类固醇生物合成的酶通常在底物结合和催化方面非常特异。然而，这些不同形式控制这种混杂性或特异性的分子机制还不清楚。结构是如何编码这种多样性的？已知在底物识别过程中，许多（如果不是全部）P450发生显著的构象变化。这些变化也可能在门控O2活化后的反应中发挥作用，以产生活性中间体。然而，我们对底物如何被特定的P450识别或这些变化如何与功能结合知之甚少。P450是通过诱导拟合还是通过仅动态采样少数优选构象来识别其底物？在过去的一段时间里，结果产生了显着的见解底物结合P450后发生的构象变化，他们建议这些变化可能是如何耦合到功能，他们提供了一个平台，开发新的催化剂与设计的特异性。目前的目标将使用P450活性位点的分子探针库来控制拟议的活性中间体的形成和性质。这些研究将测试特定的假设，根据我们最近的进展，关于活性位点的构象变化如何有助于控制O2活化步骤。我们将开发这些方法来研究专门连接到电极表面的P450的电化学行为。最后，我们将探讨使用特定的探针的分子进化的新型催化剂与设计的底物特异性的潜力。这些研究将有助于更好地理解这些重要酶的蛋白质结构如何与功能和底物特异性相结合。

DESCRIPTION (provided by applicant): Understanding the diverse chemistry displayed by P450s is a central issue in biomedical science and requires understanding their notorious propensity for specificity and promiscuity. Drug metabolizing P450s display a remarkable promiscuity in substrate recognition, while enzymes involved in steroid biosynthesis are often very specific with respect to substrate binding and catalysis. However, the molecular mechanism by which these different forms control this promiscuity or specificity is not well understood. How does structure encode such diversity? It is known that significant conformational changes occur in many, if not all, P450s during substrate recognition. These changes may also play a role in gating the reactions following O2 activation to produce reactive intermediates. However, we know very little about how substrates are recognized by a particular P450 or how these changes are coupled to function. Does a P450 recognize its substrate by induced fit or by dynamically sampling only a few preferred conformations? Results in the past period have produced significant insights into the conformational changes that occur upon substrate binding to P450, they suggest how these changes may be coupled to function, and they provide a platform for development of novel catalysts with designed specificity. The current aims will use a library of molecular probes for the P450 active site to control the formation and properties of proposed reactive intermediates. These studies will test specific hypotheses, based on our recent progress, about how conformational changes at the active site may help control the O2 activation step. We will develop these approaches to investigate the electrochemical behavior of P450s specifically wired to electrode surfaces. Finally, we will explore the potential for using specific probes for molecular evolution of novel catalysts with designed substrate specificity. These studies will contribute to a better understanding how the protein structure of these important enzymes is coupled to function and substrate specificity.