ABCC8 p.Lys880Ala
| Predicted by SNAP2: | A: D (59%), C: N (61%), D: N (61%), E: N (53%), F: D (66%), G: N (66%), H: N (66%), I: D (63%), L: D (66%), M: D (59%), N: N (82%), P: D (75%), Q: N (72%), R: N (82%), S: N (78%), T: D (59%), V: D (63%), W: D (71%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D, |
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[hide] The appended C-domain of human methionyl-tRNA synt... Biochemistry. 2001 Nov 27;40(47):14309-16. Kaminska M, Shalak V, Mirande M
The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.
Biochemistry. 2001 Nov 27;40(47):14309-16., [PMID:11714285]
Abstract [show]
An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.
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No. Sentence Comment
53 Site-directed mutagenesis of Arg857, Lys860, Lys863, Lys866, and Lys880 into Ala was performed according to the method of Ho et al. (25).
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ABCC8 p.Lys880Ala 11714285:53:65
status: NEW125 Table 1: Apparent Dissociation Constants of Wild-Type and Mutant Human MetRS for tRNAMet and Acc-tRNAMet Determined by a Gel Retardation Assay Kd for tRNAMet (µM) Kd for Acc-tRNAMet (µM) MetRS 0.1 0.5 MetRS-∆K 1.5 ~10.0 MetRS-∆C 4.0 ~10.0 MetRS-R857A 0.4 1.5 MetRS-K860A 1.5 8.0 MetRS-K863A 0.15 0.5 MetRS-K866A 0.15 0.5 MetRS-K880A 2.5 8.0 a Standard errors for Kd are in the range of 20-30% of the value.
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ABCC8 p.Lys880Ala 11714285:125:351
status: NEW150 Table 2: Apparent Kinetic Parametersa for the tRNAMet Aminoacylation Reactionb of Rabbit Elongator tRNAMet with Wild-Type and Mutant MetRS KM (µM) kcat (s-1) MetRS-Cxc 3.9 ( 1.3 0.46 ( 0.05 MetRS 3.5 ( 1.0 0.15 ( 0.04 MetRS-∆K 2.2 ( 0.7 0.09 ( 0.02 MetRS-∆C 32 ( 4 2.4 ( 0.5 MetRS-R857A 5.7 ( 1.1 0.47 ( 0.05 MetRS-K860A 17.2 ( 5.0 0.85 ( 0.15 MetRS-K863A 3.3 ( 0.8 0.22 ( 0.03 MetRS-K866A 3.9 ( 1.4 0.23 ( 0.04 MetRS-K880A 16.3 ( 5.7 1.03 ( 0.20 a Standard errors were determined from at least two independent data sets.
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ABCC8 p.Lys880Ala 11714285:150:437
status: NEW160 We substituted one by one these basic residues with Ala to give the R857A, K860A, K863A, K866A, and K880A mutants of human MetRS.
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ABCC8 p.Lys880Ala 11714285:160:100
status: NEW163 Using the gel-mobility shift assay described above, mutants K860A and K880A displayed a large decrease in their affinity for tRNAMet and Acc-tRNAMet (15-25-fold; Table 1).
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ABCC8 p.Lys880Ala 11714285:163:70
status: NEW169 The KM and kcat values determined for MetRS-K863A and -K866A were similar to those of the wild type (Table 2), and both values were significantly higher for mutants K860A and K880A.
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ABCC8 p.Lys880Ala 11714285:169:175
status: NEW