期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7954-7959
DOI：10.1073/pnas.1506792112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceDuring the past 30 years, the methyl transfer community has attempted to find the molecular origin of the methyltransferases' catalytic power. This report describes a combination of experimental and computational studies of enzymatic methyl transfer catalyzed by catechol-O-methyltransferase and its mutants at position Tyr68. The results show structural and dynamical differences between WT and mutants, as well as a role for substrate ionization in the generation of active site compaction. For the first time, to our knowledge, we are able to show a trend in donor-acceptor distance in the ground state that can be correlated with catalytic efficiency. This work provides an important step forward and a clear new direction for understanding enzymatic methyl transfer. 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.
