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首页> 外文期刊>Biochemistry >AN ACTIVE SITE PHENYLALANINE OF 3-OXO-DELTA(5)-STEROID ISOMERASE IS CATALYTICALLY IMPORTANT FOR PROTON TRANSFER
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AN ACTIVE SITE PHENYLALANINE OF 3-OXO-DELTA(5)-STEROID ISOMERASE IS CATALYTICALLY IMPORTANT FOR PROTON TRANSFER

机译：活性的3-OXO-DELTA（5）-甾体异构酶苯丙氨酸对质子转移具有催化作用

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摘要

3-Oxo-Delta(5)-steroid isomerase (KSI) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4)-isomers through the intermediate formation of a dienolate ion. This dienolate is formed by proton transfer from C-4 of the substrate to Asp-38, which then protonates the dienolate at C-6. Catalysis is enhanced by electrophilic assistance (hydrogen bonding) to the 3-oxygen by Tyr-14. We have investigated the effect of modifying phenylalanine-101 (F101), a hydrophobic residue that is located in the binding pocket of KSI. Two mutant enzymes (F101L and F101A) of KSI were prepared, and their kinetic properties were examined with 5-androstene-3,17-dione (1) as the substrate. Both of the mutants show reduced values of k(cat) compared to the wild type (WT), by about 30-fold (F101L) and by 270-fold (F101A), with only a small difference in K-m values. There is Little change in the K-i's (less than or equal to 4-fold) for the product 4-androstene-3,17-dione (3), although both enzymes bind the intermediate analog d-equilenin (4) about 25-fold less tightly than does the WT. Fluorescence spectra of 4 bound to each of these enzymes suggest that 4 is ionized at the active site of WT, un-ionized at the active site of F101A and a mixture of these ionization states at the active site of F101L. Free energy profiles are constructed for each of the mutant enzymes, and these are compared to the free energy profile for the WT. The results are interpreted in terms of stabilization of the intermediate dienolate and the flanking transition states by the phenyl ring of F101.
机译：3-氧代-三角洲（5）-类固醇异构酶（KSI）从假单胞菌睾丸激素催化各种3-氧代-三角洲（5）-类固醇通过二烯酸酯的中间形成异构化为其共轭的三角洲（4）-异构体离子。通过从底物的C-4质子转移到Asp-38形成二烯酸酯，然后在C-6处使二烯酸酯质子化。 Tyr-14通过亲电子辅助（氢键）增强了对3-氧的催化作用。我们已经研究了修饰苯丙氨酸101（F101）的作用，苯丙氨酸101是位于KSI结合口袋中的疏水残基。制备了KSI的两种突变酶（F101L和F101A），并以5-雄烯-3,17-二酮（1）为底物检测了它们的动力学性质。与野生型（WT）相比，这两个突变体均显示k（cat）值降低了30倍（F101L）和270倍（F101A），而K-m值只有很小的差异。产物4-雄甾烯-3,17-二酮（3）的Ki's变化很小（小于或等于4倍），尽管两种酶都结合中间类似物d-quilenin（4）约25 -比WT折叠得不紧。与每种酶结合的4的荧光光谱表明4在WT的活性位点被电离，在F101A的活性位点被非电离，并且在F101L的活性位点上这些电离态的混合物。为每种突变酶构建自由能谱，并将其与野生型的自由能谱进行比较。用F101的苯环稳定中间二烯酸酯和侧接过渡态来解释结果。

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