title: Single-Turnover Kinetics of Methyl Transfer to tRNA by Methyltransferases.
abstract: Methyl transfer from S-adenosyl methionine (abbreviated as AdoMet) to biologically active molecules such as mRNAs and tRNAs is one of the most fundamental and widespread reactions in nature, occurring in all three domains of life. The measurement of kinetic constants of AdoMet-dependent methyl transfer is therefore important for understanding the reaction mechanism in the context of biology. When kinetic constants of methyl transfer are measured in steady state over multiple rounds of turnover, the meaning of these constants is difficult to define and is often limited by non-chemical steps of the reaction, such as product release after each turnover. Here, the measurement of kinetic constants of methyl transfer by tRNA methyltransferases in rapid equilibrium binding condition for one methyl transfer is described. The advantage of such a measurement is that the meaning of kinetic constants can be directly assigned to the steps associated with the chemistry of methyl transfer, including the substrate binding affinity to the methyltransferase, the pre-chemistry re-arrangement of the active site, and the chemical step of methyl transfer. An additional advantage is that kinetic constants measured for one methyl transfer can be correlated with structural information of the methyltransferase to gain direct insight into its reaction mechanism.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, BLSB 220, Philadelphia, PA, 19107-5719, USA. ya-ming.hou@jefferson.edu.
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; C489297:TrmD protein, E coli; D012359:tRNA Methyltransferases
keywords: AdoMet-dependent methyl transfer; Rapid equilibrium binding