| RRC ID |
35372
|
| 著者 |
Pearson D, Carell T.
|
| タイトル |
Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.
|
| ジャーナル |
Nucleic Acids Res
|
| 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.
|
| 巻・号 |
39(11)
|
| ページ |
4818-26
|
| 公開日 |
2011-6-1
|
| DOI |
10.1093/nar/gkr071
|
| PII |
gkr071
|
| PMID |
21306992
|
| PMC |
PMC3113582
|
| MeSH |
Chromatography, High Pressure Liquid / methods
Escherichia coli Proteins / metabolism*
Kinetics
Multienzyme Complexes / metabolism*
RNA, Transfer, Glu / chemistry
RNA, Transfer, Glu / metabolism*
Thiouridine / analogs & derivatives*
Thiouridine / metabolism
|
| IF |
11.502
|
| 引用数 |
11
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
ME9062(BW25113)
JW5380-KC |
