A link between protein structure and enzyme catalyzed hydrogen tunneling
A link between protein structure and enzyme catalyzed hydrogen tunneling
复制标题
DOI：
10.1073/pnas.94.24.12797
复制
发表时间：
1997-11-25
影响因子：
11.1
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Bahnson, BJ;Colby, TD;Klinman, JP
我们提出的证据表明，活性中心侧链的大小可以调节酶催化反应中氢隧道的程度，已经测量了25℃下马肝醇脱氢酶催化苯甲醇氧化的一级和二级k(H)/k(T)和k(D)/k(T)动力学同位素效应，正如早期研究报告的那样，二级k(H)/k(T)和k(D)/k(T)同位素效应之间的关系为偏离经典行为提供了灵敏的探针。在本工作中，对乙醇脱氢酶催化的203位点突变之间的氢气转移的催化效率和氢隧道的程度进行了关联。VAL-203与乙醇底物辅因子NAD(+)的反面相互作用，残基尺寸的减小与隧道效应的减少和催化效率的两个数量级的下降相关。比较了高隧道(Phe-93-->；Trp)和低隧道(Val-203->；Ala)突变体的三元配合物的X射线晶体结构，为观察到的效应提供了结构基础，证明了低隧道突变体的氢转移距离增加，Val-203-->；三元配合物晶体结构也显示出相对于野生型和Phe-93->；的超封闭的域间几何结构；Trp突变的三元复杂结构，这表明了域间移动的灵活性，这可能会缩短天然酶中供体和受体碳之间的距离，并可能增强隧道效应在氢化物转移反应中的作用。
We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction, Primary and secondary k(H)/k(T) and k(D)/k(T) kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the relationship between secondary k(H)/k(T) and k(D)/k(T) isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD(+) from the alcohol substrate, The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 --> Trp) and a low-tunneling (Val-203 --> Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant, The Val-203 --> Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 --> Trp mutant ternary complex structures, This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.