Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
J Inorg Biochem. 2010 Nov;104(11):1185-94. doi: 10.1016/j.jinorgbio.2010.07.008. Epub 2010 Jul 23.
The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25°C. The activity constants (k(cat)) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25° C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70° C. The rate of catalysis at temperatures above ~ 70° C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.
设计了嗜热 P450（P450 175A1 或 CYP175A1）的位点特异性突变体，以引入靠近活性位点的可以充当酸碱催化剂的残基，从而增强过氧化物酶的活性。CYP175A1 远端血红素口袋中的亮氨酸 80 位于与参与稳定氯化过氧化物酶（CPO）中 ferryl 血红素中间体的谷氨酸 183 几乎等效的位置。CYP175A1 的亮氨酸 80 残基被组氨酸（L80H）和谷氨酰胺（L80Q）突变，这两种残基可能与过氧化氢形成氢键，并促进过氧化物酶循环中假定的氧化还原中间产物的形成和稳定。与野生型（WT）CYP175A1 酶相比，突变体 L80H 和 L80Q 的过氧化物酶活性在 25°C 下更高。在 25°C 下，L80H 和 L80Q 突变体的 CYP175A1 的活性常数（kcat）高于肌红蛋白和野生型细胞色素 b562。WT 和 CYP175A1 突变体过氧化物酶活性的最佳温度约为 70°C。在高于约 70°C 的温度下，L80Q 突变体的催化速率高于著名的天然过氧化物酶辣根过氧化物酶（HRP），因为 HRP 在如此高的温度下会变性。与 HRP 不同，CYP175A1 突变体的过氧化物酶活性在 pH 9 时达到最大值，HRP 的过氧化物酶活性在 pH~5 时达到最大值。这些结果已经根据对这些热稳定血红素蛋白过氧化物酶性质的结构-功能关系的理解进行了讨论。
