Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole
Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole
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DOI：
10.1073/pnas.0911168107
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发表时间：
2010-02-02
影响因子：
11.1
通讯作者：
Herschlag, Daniel
中科院分区：
文献类型：
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作者：
Kraut, Daniel A.;Sigala, Paul A.;Herschlag, Daniel
酶活性位点相互作用的催化重要性经常通过突变特定残基和测量所产生的速率降低来评估。这种方法已用于细菌甾酮异构酶中，以探测贡献给二烯醇化物反应中间体的活性位点氢键的能量重要性。保守的Tyr 16 Phe突变使催化作用减弱10（5）倍，远大于其他酶中氢键突变的影响。然而，保守性较低的Tyr 16 Ser突变也会干扰Tyr 16氢键，导致不太严重的10（2）倍速率降低。为了理解这些突变的矛盾效应，并澄清Tyr 16氢键的能量重要性，我们已经确定了1.6埃分辨率的X-射线结构的中间类似物，equilenin，绑定到Tyr 16 Ser突变体和测量率的影响突变Tyr 16丝氨酸，苏氨酸，丙氨酸，甘氨酸。这些突变的几乎相同的200倍速率降低，以及在X射线结构中观察到的Ser 16羟基和马烯雌酚氧之间的6.4埃距离，强烈表明该突变体的更温和的速率效应不是由于在位置16处维持来自Ser的氢键。这些结果，额外的光谱观察，和以前的结构研究表明，Tyr 16 Phe突变的结果在不利的相互作用与二烯醇中间体以外的损失的氢键，从而夸大了明显的能量效益的Tyr 16氢键相对于溶液反应。这些结果强调了氢键相互作用和定点诱变实验的复杂能量学。
The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 10(5)-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 10(2)-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-angstrom resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-angstrom distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and sitedirected mutagenesis experiments.