Zawrotny M E, Pollack R M
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228-5398.
Biochemistry. 1994 Nov 22;33(46):13896-902. doi: 10.1021/bi00250a044.
3-Oxo-delta 5-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated delta 4-isomers through the formation of an intermediate dienol. Mutation of the catalytic base (Asp-38) to Glu (D38E) has been found to reduce kcat/Km for the isomerization of 5-androstene-3,-17-dione (1) to 4-androstene-3,17-dione (3) by about 300-fold (Zawrotny et al., 1991). The free energy profile for the D38E enzyme was determined from a combination of steady state kinetics and stopped-flow kinetics with the independently generated dienol intermediate (2). A comparison of the energetics of D38E with that of the wild type enzyme (WT) shows that the only significant difference is a reduction in the rates of the chemical steps for the interconversion of 1, 2, and 3 on the enzyme surface by about 10(3)-fold for D38E. The relative energy levels for all bound species are nearly identical for WT and D38E, whereas the transition states for both enolization and ketonization are destabilized by 3-4 kcal/mol. The effect of the D38E mutation on the energetics of KSI is comparable to the corresponding effect of the E165D mutation on the energetics of triosephosphate isomerase (TIM).
3-氧代-δ5-类固醇异构酶（KSI）通过形成中间体烯醇，催化多种3-氧代-δ5-类固醇异构化为其共轭δ4-异构体。已发现催化碱基（Asp-38）突变为Glu（D38E）会使5-雄烯-3,17-二酮（1）异构化为4-雄烯-3,17-二酮（3）的kcat/Km降低约300倍（扎罗特尼等人，1991年）。D38E酶的自由能分布是通过稳态动力学和停流动力学与独立生成的烯醇中间体（2）相结合来确定的。D38E与野生型酶（WT）的能量学比较表明，唯一显著的差异是D38E使酶表面上1、2和3相互转化的化学步骤速率降低了约10³倍。WT和D38E所有结合物种的相对能量水平几乎相同，而烯醇化和酮化的过渡态都不稳定了3-4千卡/摩尔。D38E突变对KSI能量学的影响与E165D突变对磷酸丙糖异构酶（TIM）能量学的相应影响相当。
