Pollack R M, Thornburg L D, Wu Z R, Summers M F
Department of Chemistry, Howard Hughes Medical Institute, University of Maryland Baltimore County, Baltimore, Maryland, 21250, USA.
Arch Biochem Biophys. 1999 Oct 1;370(1):9-15. doi: 10.1006/abbi.1999.1384.
3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through the formation of an intermediate dienolate. The three-dimensional structure of the enzyme from Pseudomonas testosteroni was solved by multidimensional heteronuclear magnetic resonance spectroscopy. This protein, a 28-kDa symmetric dimer, exhibits a three-dimensional fold with the two independently folded monomers packed together via extensive hydrophobic and electrostatic interactions. The previously identified catalytically important residues Tyr-14 (general acid) and Asp-38 (general base) are located near the bottom of a deep hydrophobic cavity and are positioned in a manner consistent with previous mechanistic hypotheses. The structure also revealed the presence of an unexpected acid group (Asp-99) located in the active site adjacent to Tyr-14. Mutagenesis and kinetic studies show that Asp-99 has an anomalously high pK(a) (>9), which allows it to contribute to catalysis by donating a hydrogen bond to the intermediate and to the transition states. In support of this hypothesis, effects on the kinetic parameters of the mutations Y14F and D99A are additive in the Y14F/D99A mutant.
3-氧代-δ(5)-类固醇异构酶（KSI）通过形成中间体烯醇化物催化β,γ-不饱和3-氧代类固醇异构化为其共轭异构体。通过多维异核磁共振光谱解析了睾丸酮假单胞菌中该酶的三维结构。这种蛋白质是一种28 kDa的对称二聚体，呈现出一种三维折叠结构，两个独立折叠的单体通过广泛的疏水和静电相互作用堆积在一起。先前确定的具有催化重要性的残基Tyr-14（一般酸）和Asp-38（一般碱）位于一个深疏水腔的底部附近，其定位方式与先前的机理假设一致。该结构还揭示了在与Tyr-14相邻的活性位点存在一个意外的酸性基团（Asp-99）。诱变和动力学研究表明，Asp-99具有异常高的pK(a)（>9），这使其能够通过向中间体和过渡态提供氢键来促进催化作用。为支持这一假设，在Y14F/D99A突变体中，对Y14F和D99A突变的动力学参数的影响是累加的。
