Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase
Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase
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DOI：
10.1529/biophysj.107.121913
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发表时间：
2008-05-15
影响因子：
3.4
通讯作者：
Schwartz, Steven D.
中科院分区：
文献类型：
--
作者：
Saen-Oon, Suwipa;Ghanem, Mahmoud;Schwartz, Steven D.
研究发现，在人嘌呤核苷磷酸化酶（hPNP）的两个表面残基(Lys（22) -> Glu和His(104) -> Arg）发生突变后，该化学步骤的催化活性增强。这是正确的，尽管突变离活性位点相当远，并且在野生型和突变型活性位点之间没有明显的晶体结构变化。我们认为，从远端残基到催化位点的动态耦合可能在催化中起作用，正是这种动力学的改变导致了化学步骤速率的增加。计算结果表明，突变体在促进振动和反应坐标之间的耦合比天然hPNP更强。比较原生蛋白和突变蛋白的功率谱显示Immucillin-G (ImmG):O5‘中心点中心点ImmG:N4’和H257: N δ中心点中心点中心点ImmG:O5'的振动之间存在相关性，符合这些运动的耦合。这些模式与促进振动的蛋白质有关。运动与反应坐标的强耦合增加了达到过渡态的概率，从而降低了激活自由能。这一运动已被证明有助于催化作用。随着过渡态的接近，ImmG: o4 ‘中心点中心点中心点ImmG: o5 ’中心点中心点中心点H257:N δ的距离之和变小，稳定了过渡态形成的氧碳离子。晶体学、突变分析、化学动力学和计算分析的综合结果表明，动态压缩在过渡态的形成中起着重要作用。在催化增强的突变酶中观察到这些对更强的偶联。远离催化位点的突变增强了运动和催化作用，这暗示了通过蛋白质结构作为hPNP催化成分的动态偶联。
It has been found that with mutation of two surface residues (Lys(22) -> Glu and His(104) -> Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5'center dot center dot center dot ImmG:N4' and H257: N delta center dot center dot center dot ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'center dot center dot center dot ImmG:O5'center dot center dot center dot H257:N delta became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.