Hughes Richard K, Belfield Eric J, Muthusamay Mylrajan, Khan Anuja, Rowe Arthur, Harding Stephen E, Fairhurst Shirley A, Bornemann Stephen, Ashton Ruth, Thorneley Roger N F, Casey Rod
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Biochem J. 2006 May 1;395(3):641-52. doi: 10.1042/BJ20051667.
We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.
我们描述了来自蒺藜苜蓿（桶状苜蓿）的具有氢过氧化物裂解酶（HPL）活性的重组植物细胞色素P450酶CYP74C3（细胞色素P450亚家族74C3）的详细生化特性。报告了稳态动力学参数、底物和产物特异性、RZ（纯度指数）、摩尔吸收系数、血红素含量以及HPL的新配体。我们基于凝胶过滤、沉降速度（沉降系数分布）和沉降平衡（分子量）分析表明，CYP74C3作为一种无洗涤剂、水溶性的单体，酶活性较低。用洗涤剂胶束而非洗涤剂单体重新激活可完全恢复酶活性。自旋态平衡的相应变化以及可能的血红素铁配位，对于细胞色素P450酶来说是新颖的，这表明洗涤剂胶束对蛋白质构象有微妙影响，而非底物呈现，这足以将底物结合和催化中心活性提高一个数量级。高达1.6×10⁸ M⁻¹×s⁻¹的kcat/K(m)是记录到的最高值之一，对于一种反应机制涉及碳 - 碳键断裂的酶来说，这是非常显著的。我们进行了动力学和生物物理研究，以证明这种效应是蛋白质单体与单个洗涤剂胶束形成复合物的结果。与洗涤剂胶束结合而非寡聚状态代表了膜相关细胞色素P450酶激活的一种新机制。高度浓缩且单分散的无洗涤剂CYP74C3蛋白样品可能非常适合用于第一种植物细胞色素P450酶的结晶和结构解析。
