Puchkaev Andrei V, Koo Laura S, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Arch Biochem Biophys. 2003 Jan 1;409(1):52-8. doi: 10.1016/s0003-9861(02)00402-2.
Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.
研究了嗜热细胞色素P450 119（CYP119）的两个显著特征，即F-G环与I螺旋之间的Arg154-Glu212盐桥和一个延伸的芳香簇，以确定它们对该酶热稳定性的贡献。表达并纯化了盐桥（Arg154、Glu212）和芳香簇（Tyr2、Trp4、Trp231、Tyr250、Trp281）的位点特异性突变体。大多数突变体中月桂酸羟基化的底物结合常数和动力学常数受影响较小，但E212D突变体无活性，R154Q突变体具有更高的K(s)、K(m)和k(cat)值。盐桥突变体与野生型CYP119一样，在91±1℃时熔化，而延伸芳香簇中单个残基的突变使熔点降低10-15℃，尽管在一个不相关的芳香残基发生突变时未观察到变化。延伸的芳香簇而非Arg154-Glu212盐桥对CYP119的热稳定性有贡献。
