title: Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.
abstract: Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.
authors.affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Pearson
authors.forename: Thomas
authors.initials: T
authors.lastname: Carell
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D012352:RNA, Transfer, Glu / Q000737:chemistry / Q000378:metabolism*; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C488767:MnmC protein, E coli; D009097:Multienzyme Complexes; D012352:RNA, Transfer, Glu; D013891:Thiouridine; C011916:5-methylaminomethyl-2-thiouridine; C030546:5-(carboxymethylaminomethyl)-2-thiouridine