Elucidation for the catalytic function of cytochrome P450cam by site-specific incorporation of natural and unnatural amino acid.

细胞色素P450cam （Cytochrome P450cam）是一种含血红素的单加氧酶，可催化d-樟脑+ NADH + H^+ + O_2 * 5-外羟基樟脑+ H_2O + NAD^+的反应。在这个反应中，NADH的2个电子通过聚合还原酶和聚合还原蛋白（Pdx）传递到P450cam。P450cam在其处于铁态和随后的氧-铁态的步骤中被Pdx还原。在两年的项目中，我们发现P450cam中的一种表面残留物Arg112对第一和第二还原步骤至关重要。动力学研究表明：1)Arg112是Pdx的结合位点，2)Arg112是分子内电子转移（Pdx-P450cam）的重要残基，也是控制P450cam血红素片段氧化还原电位的重要残基。本项目的另一个重要成果是我们成功地将位点特异性非天然氨基酸0-甲基苏氨酸掺入P450cam的112位。对该突变体的催化活性测定表明，该突变体酶的耗氧活性仅为野生型酶的三分之一，但将消耗的分子二氧的氧原子全部吸收到d-樟脑的5-外显位。这表明苏氨酸的羟基在单氧反应中并不是必不可少的。我们之前的研究表明，只有当羟基位于P450cam的112位时，d-樟脑的单氧合才能有效地完成。基于这些和其他结果，我们提出了质子供体和/或酸催化作为Thr252的作用。为了验证Thr的这一作用，我们计划了这个项目，并成功地展示了非天然氨基酸的位点特异性掺入对阐明酶的催化机制的重要性。

Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the reaction : d-camphor + NADH + H^+ + O_2 * 5-exo-hydroxycamphor + H_2O + NAD^+. In this reaction, 2 electrons from NADH are deliveredto P450cam via putidaredoxinreductaseand putidaredoxin (Pdx). P450cam is reduced by Pdx at the step where it is in the ferric and subsequent oxy-ferrous states.During two years of this project, we have found that a surface residue, Arg112 in P450cam is crucial for the first and second reduction steps. Kinetic studies suggests that : 1) Arg112 forms the binding site for Pdx. 2) Arg112 is an important residue for intramolecular electron transfer (Pdx-P450cam) and also for controlling redox potentials of the P450cam heme-moiety. Another important results of this project is that we have successfully incorporated site-specifically unnatural amino acid, 0-methyl-threonine to the 112 position of P450cam. Catalytic activity measurement of this mutant shows that the mutant enzyme incorporates all the oxygen atom of molecular dioxygen consumed to 5-exo-position of d-camphor, although oxygen consuming activity is one third of that of the wild-type enzyme. This results indicate that the hydroxygroup of threonine is not indispensable for the monooxygenation reaction.We previously showed that monooxygnation of d-camphor was only accomplished efficiently when the hydroxygroup is at 112 position of P450cam. Based on these and other results, we proposed a proton donor and/or acid catalysis as a role of Thr252. In order to validate this proposed role of Thr, we have planned this project and showed successfully the importance of the site-specific incorporation of unnatural amino acid to elucidate the catalytic mechanism of the enzyme.