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

Pearson D, Carell T - Nucleic Acids Res. (2011)

Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

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.

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Biosynthesis of mnm5(s2)U in tRNA. X = O (in U) or S (in s2U), R = cmnm5, nm5 or mnm5. Modified positions are coloured in red.
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SCH1: Biosynthesis of mnm5(s2)U in tRNA. X = O (in U) or S (in s2U), R = cmnm5, nm5 or mnm5. Modified positions are coloured in red.


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

Pearson D, Carell T - Nucleic Acids Res. (2011)

Biosynthesis of mnm5(s2)U in tRNA. X = O (in U) or S (in s2U), R = cmnm5, nm5 or mnm5. Modified positions are coloured in red.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113582&req=5

SCH1: Biosynthesis of mnm5(s2)U in tRNA. X = O (in U) or S (in s2U), R = cmnm5, nm5 or mnm5. Modified positions are coloured in red.
Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

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.

Show MeSH
Related in: MedlinePlus