ABCMdb

PMID: 11714285

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., [PubMed]

Sentences

No. Mutations Sentence Comment

53 ABCC8 p.Lys880Ala Site-directed mutagenesis of Arg857, Lys860, Lys863, Lys866, and Lys880 into Ala was performed according to the method of Ho et al. (25). Login to comment 125 ABCC8 p.Lys880Ala 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. Login to comment 150 ABCC8 p.Lys880Ala 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. Login to comment 160 ABCC8 p.Lys880Ala We substituted one by one these basic residues with Ala to give the R857A, K860A, K863A, K866A, and K880A mutants of human MetRS. Login to comment 163 ABCC8 p.Lys880Ala 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). Login to comment 169 ABCC8 p.Lys880Ala 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. Login to comment