酶促甲基转移反应中供体-受体距离的介导作用。
Mediation of donor-acceptor distance in an enzymatic methyl transfer reaction.
作者信息
Zhang Jianyu, Kulik Heather J, Martinez Todd J, Klinman Judith P
机构信息
Department of Chemistry, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
Department of Chemistry, University of California, Berkeley, CA 94720; Photon Ultrafast Laser Science and Engineering Institute and Department of Chemistry, Stanford University, Stanford, CA 94305;
出版信息
Proc Natl Acad Sci U S A. 2015 Jun 30;112(26):7954-9. doi: 10.1073/pnas.1506792112. Epub 2015 Jun 15.
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.
利用结合同位素效应（BIEs）、时间分辨荧光寿命、斯托克斯位移以及基于扩展图形处理单元（GPU）的量子力学/分子力学（QM/MM）方法，对由儿茶酚-O-甲基转移酶（COMT）催化的酶促甲基转移进行了研究。将野生型酶与位于辅因子反应性硫原子后方的保守残基Tyr68处的突变体进行了比较。在含有8-羟基喹啉的S-腺苷甲硫氨酸（AdoMet）二元和流产三元复合物中观察到了较大（>1）的BIEs，这与先前报道的反向（<1）动力学同位素效应（KIEs）形成对比。对含有儿茶酚盐的三元复合物进行的基于GPU的扩展计算研究表明，基态结构存在明显趋势，从野生型的非标准键长向突变体的溶液值转变。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在活性位点压缩的背景下进行了讨论，活性位点压缩需要酶三元复合物内底物的电离。
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