Mediation of donor-acceptor distance in an enzymatic methyl transfer reaction.
Mediation of donor-acceptor distance in an enzymatic methyl transfer reaction.
复制标题
酶促甲基转移反应中供体-受体距离的调节。
DOI：
10.1073/pnas.1506792112
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发表时间：
2015
影响因子：
11.1
通讯作者：
Klinman,JudithP
中科院分区：
文献类型：
--
作者：
Zhang,Jianyu;Kulik,HeatherJ;Martinez,ToddJ;Klinman,JudithP
用结合同位素效应(Bie)、时间分辨荧光寿命、斯托克斯位移和基于扩展图形处理单元(GPU)的量子力学/分子力学(QM/MM)方法研究了邻苯二酚-O-甲基转移酶(COMT)催化的酶促甲基转移。WT酶与突变体在Tyr68上进行了比较，Tyr68是一个保守残基，位于辅因子的活性硫之后。在含有8-羟基喹啉的S-腺苷甲硫氨酸(ADOMet)-二元和流产三元络合物中观察到了小的(>；1)Bie，并与先前报道的(<；1)动力学同位素效应(Kie)进行了对比。基于GPU的扩展计算研究表明，含有儿茶酸酯的三元络合物的基态结构有明显的趋势，从WT的非正则键长到含有突变体的溶液的值。使用时间分辨斯托克斯位移测量和分子动力学也检测到了对Tyr68敏感的结构和动力学差异。这些实验和计算结果是在活性中心紧凑的背景下讨论的，这需要酶三元复合体中的底物电离。
Enzymatic methyl transfer, catalyzed by catechol-O-methyltransferase (COMT), is investigated using binding isotope effects (BIEs), time-resolved fluorescence lifetimes, Stokes shifts, and extended graphics processing unit (GPU)-based quantum mechanics/molecular mechanics (QM/MM) approaches. The WT enzyme is compared with mutants at Tyr68, a conserved residue that is located behind the reactive sulfur of cofactor. Small (>1) BIEs are observed for an S-adenosylmethionine (AdoMet)-binary and abortive ternary complex containing 8-hydroxyquinoline, and contrast with previously reported inverse (<1) kinetic isotope effects (KIEs). Extended GPU-based computational studies of a ternary complex containing catecholate show a clear trend in ground state structures, from noncanonical bond lengths for WT toward solution values with mutants. Structural and dynamical differences that are sensitive to Tyr68 have also been detected using time-resolved Stokes shift measurements and molecular dynamics. These experimental and computational results are discussed in the context of active site compaction that requires an ionization of substrate within the enzyme ternary complex.