Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, California 95616, USA.
J Am Chem Soc. 2013 Feb 20;135(7):2509-11. doi: 10.1021/ja3101243. Epub 2013 Feb 5.
The catalytic effects of perdeuterating the pyridoxal phosphate-dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The mass of the heavy perdeuterated form is ~5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of ~1.3 on k(cat) and k(cat)/K(M) for both L- and D-alanine. These values increase when Cα-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of ~3 on k(cat)/K(M) with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KIEs. This breakdown of the rule of the geometric mean is likely due to coupled motion between the protein and the proton-transfer reaction coordinate in the rate-limiting step. These data implicate a direct role for protein vibrational motions in barrier crossing for proton-transfer steps in alanine racemase.
报道了来自嗜热脂肪芽孢杆菌的吡哆醛磷酸依赖酶丙氨酸消旋酶的去氘化的催化作用。重氘化形式的质量比氕化形式大约大 5.5%，导致 L-和 D-丙氨酸的 k(cat)和 k(cat)/K(M)的动力学同位素效应（KIEs）分别约为 1.3。当使用 Cα-氘代丙氨酸作为底物时，这些值会增加。重酶的 KIEs 与氘代底物的 k(cat)/K(M)分别约为 3，大于单个重酶和初始底物 KIEs 的乘积。这种打破几何平均值规则的情况可能是由于在限速步骤中蛋白质和质子转移反应坐标之间的耦合运动所致。这些数据表明，在丙氨酸消旋酶的质子转移步骤中，蛋白质振动运动在势垒穿越中起直接作用。
