Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, CT 06520, USA.
Science. 2013 Aug 23;341(6148):899-903. doi: 10.1126/science.1241735.
Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using nuclear magnetic resonance spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates; however, we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes.
许多研究表明，构象运动在催化循环中起作用，作用是优化结合口袋或促进产物释放，但在化学反应中发挥更密切的作用尚未被描述。我们通过使用核磁共振波谱法监测两种蛋白酪氨酸磷酸酶（PTPs）中的活性位点环运动来解决这个问题。这两种 PTPs，YopH 和 PTP1B，具有非常不同的催化速率；然而，我们发现它们的活性位点环都以与其磷酸酪氨酸切割动力学相匹配的速率关闭到其催化有效位置。这个环包含催化酸，这表明环的关闭与离去基团酪氨酸的质子化同时发生，这解释了两种在化学和机械上难以区分的酶的不同动力学。
