Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 02/2011; 39(11):4818-26. DOI: 10.1093/nar/gkr071
Source: PubMed


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.


Available from: Thomas Carell, Apr 21, 2015

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Article: Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U

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    • "Therefore, MnmC2 may be required to convert nm5U34 not originating from MnmC1. Consistent with this notion are recent kinetic studies demonstrating that the activities of the MnmC1 and MnmC2 domains are independent of one another [13]. The lack of coupling between the two active sites in bifunctional MnmC likely ensures comparable turn over of nm5U34 generated from either MnmE/MnmG or MnmC1. "
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