Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase
Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase
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DOI：
10.1073/pnas.1704786114
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发表时间：
2017-06-20
影响因子：
11.1
通讯作者：
Schramm, Vern L.
中科院分区：
文献类型：
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作者：
Harijan, Rajesh K.;Zoi, Ioanna;Schramm, Vern L.
重酶同位素效应（N-15-，C-13-和H-2-标记的蛋白质）探索与催化有关的质量依赖性振动模式。过渡路径采样（TPS）计算预测飞秒动力学耦合在人嘌呤核苷磷酸化酶（PNP）的催化位点。在重质PNP中观察到偶联，其中缓慢的屏障穿越引起正常的重酶同位素效应（k（化学轻）/k（化学重）> 1.0）。我们使用TPS设计突变体F159 Y PNP，预测其改善重F159 Y PNP的屏障穿越，试图产生罕见的反向重酶同位素效应（k（化学轻）/k（化学重）< 1.0）。轻和重天然PNP与轻和重F159 Y PNP的稳态动力学比较揭示了相似的动力学性质。在F159 Y PNP中，前稳态化学减慢了32倍。前稳态化学比较了重和轻天然和F159 Y PNP，发现天然PNP的正常重酶同位素效应为1.31，F159 Y PNP的反向效应为0.75。F159 Y PNP中同位素质量的增加导致更有效的过渡态形成。对重F159 Y PNP的逆同位素效应的独立验证来自于对催化实验的承诺。大多数重酶表现出正常的重酶同位素效应，而F159 Y PNP是一个罕见的反向效应的例子。原生和F159 Y PNP的晶体结构和TPS动力学探索了与这些催化变化相关的催化位点几何形状。实验验证TPS预测的障碍跨越建立连接的快速蛋白质动力学和振动耦合酶的过渡态通道。
Heavy-enzyme isotope effects (N-15-, C-13-, and H-2-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femto-second dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (k(chem light)/k(chem heavy) > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (k(chem light)/k(chem heavy) < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.