Raag R, Martinis S A, Sligar S G, Poulos T L
Maryland Biotechnology Institute, University of Maryland, Shady Grove, Rockville, Maryland 20850.
Biochemistry. 1991 Dec 3;30(48):11420-9. doi: 10.1021/bi00112a008.
The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).
一种细胞色素P - 450CAM定点突变体的晶体结构已被精修至2.2埃分辨率下R因子为0.18，该突变体中活性位点苏氨酸252（Thr252）已被丙氨酸取代（Thr252Ala）。根据序列比对（尼尔森和斯特罗贝尔，1989年），苏氨酸252在P - 450酶中高度保守。亚铁结合樟脑和一氧化碳的P - 450CAM的晶体结构（拉格和普oulos，1989b）表明，苏氨酸252是关键的活性位点残基，构成双氧结合位点的一部分。活性位点苏氨酸突变为丙氨酸产生一种酶，其中底物羟基化与电子转移解偶联。具体而言，产生的是过氧化氢和“过量”的水，而不是产物5 - exo -羟基樟脑。X射线结构显示，在Gly248和Thr252之间的远端螺旋中的局部扭曲在Thr252Ala突变体中变得更加严重。此外，天然酶中不存在的一个溶剂分子位于突变酶活性位点的双氧结合区域。在这个位置，溶剂分子可能在空间上干扰并使双氧结合不稳定。此外，活性位点溶剂分子通过氢键网络与一个内部溶剂通道相连，该通道将远端螺旋残基与一个埋藏的谷氨酸侧链相连。因此，与野生型酶相比，突变体中的双氧似乎更容易接近溶剂质子，这一因素可能促进过氧化氢和/或水的产生而不是底物羟基化。基于晶体学和诱变数据，为野生型P - 450CAM提出了一条涉及残基Lys178/Arg186、Asp251和Thr252的质子传递途径。本文讨论的结构坐标已提交给布鲁克海文蛋白质数据库（伯恩斯坦等人，1977年）。
