scholarly journals Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD

1989 ◽  
Vol 258 (3) ◽  
pp. 715-721 ◽  
Author(s):  
J E C McArdell ◽  
M Duffield ◽  
T Atkinson
Keyword(s):  
Bacillus Stearothermophilus ◽  
Competitive Inhibitor ◽  
Reactive Dye ◽  
Trna Synthetase ◽  
Enzyme Inactivation ◽  
Trna Synthetases ◽  
Time Period ◽  
Substrate Binding Sites ◽  
Methionyl Trna Synthetase ◽  
Fold Excess

A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates.

scholarly journals A Novel Aminoacyl-tRNA Synthetase Appended Domain Can Supply the Core Synthetase with Its Amino Acid Substrate

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1320
Author(s):  
Marc Muraski ◽  
Emil Nilsson ◽  
Benjamin Weekley ◽  
Sandhya Bharti Sharma ◽  
Rebecca W. Alexander
Keyword(s):  
Trna Synthetase ◽  
Mycoplasma Species ◽  
Intracellular Pathogen ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Acid Analog ◽  
Methionyl Trna Synthetase ◽  
Acid Substrate ◽  
Amino Acid Substrate

The structural organization and functionality of aminoacyl-tRNA synthetases have been expanded through polypeptide additions to their core aminoacylation domain. We have identified a novel domain appended to the methionyl-tRNA synthetase (MetRS) of the intracellular pathogen Mycoplasma penetrans. Sequence analysis of this N-terminal region suggests the appended domain is an aminotransferase, which we demonstrate here. The aminotransferase domain of MpMetRS is capable of generating methionine from its α-keto acid analog, 2-keto-4-methylthiobutyrate (KMTB). The methionine thus produced can be subsequently attached to cognate tRNAMet in the MpMetRS aminoacylation domain. Genomic erosion in the Mycoplasma species has impaired many canonical biosynthetic pathways, causing them to rely on their host for numerous metabolites. It is still unclear if this bifunctional MetRS is a key part of pathogen life cycle or is a neutral consequence of the reductive evolution experienced by Mycoplasma species.

scholarly journals Nucleolar Localization of Human Methionyl–Trna Synthetase and Its Role in Ribosomal RNA Synthesis

2000 ◽  
Vol 149 (3) ◽  
pp. 567-574 ◽  
Author(s):  
Young-Gyu Ko ◽  
Young-Sun Kang ◽  
Eun-Kyoung Kim ◽  
Sang Gyu Park ◽  
Sunghoon Kim
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Trna Synthetase ◽  
Nucleolar Localization ◽  
Trna Synthetases ◽  
Quiescent Cells ◽  
Aminoacyl Trna Synthetases ◽  
Polymerase I ◽  
Methionyl Trna Synthetase

Human aminoacyl–tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl–tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.

scholarly journals Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand

1977 ◽  
Vol 167 (2) ◽  
pp. 419-428 ◽  
Author(s):  
C M Clarke ◽  
J R Knowles
Keyword(s):  
Ribonucleic Acid ◽  
Bacillus Stearothermophilus ◽  
Preceding Paper ◽  
Trna Synthetase ◽  
Rapid Preparation ◽  
Purification Method ◽  
Trna Synthetases ◽  
Trna Species ◽  
Aminoacyl Transfer ◽  
Cognate Trna

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

scholarly journals The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase

1987 ◽  
Vol 243 (3) ◽  
pp. 701-707 ◽  
Author(s):  
J E C McArdell ◽  
C J Bruton ◽  
T Atkinson
Keyword(s):  
Amino Acid ◽  
Bacillus Stearothermophilus ◽  
Catalytic Domain ◽  
Competitive Inhibitor ◽  
Trna Synthetase ◽  
Apparent Dissociation Constant ◽  
Peptide Peak ◽  
Competitive Kinetics ◽  
Amino Acid Binding ◽  
Dissociation Constant Kd

Tryptophyl-tRNA synthetase is irreversibly inactivated by Procion Brown MX-5BR with an apparent dissociation constant (KD) of 8.8 microM and maximum rate of inactivation k3 0.192 s-1. The specificity of the interaction is supported by two previously reported observations. Firstly, Brown MX-5BR inactivation of tryptophyl-tRNA synthetase is inhibited by substrates, and secondly, the animated derivative of Brown MX-5BR is a competitive inhibitor of tryptophyl-tRNA synthetase with a Ki of 2 X 10(-4) M with respect to both tryptophan and ATP. Tryptic digestion of the dye-affinity-labelled enzyme and subsequent resolution of the peptides by h.p.l.c. yielded one major dye-peptide peak. Amino acid sequence analysis resulted in the identification of the dye-binding domain centred on lysine-178. Tyrosyl-tRNA synthetase is also inactivated by Procion Brown MX-5BR, and this inactivation is prevented by ATP but not by tyrosine. The interaction of tyrosyl-tRNA synthetase with hydroxylated Brown MX-5BR exhibited non-competitive kinetics with respect to the amino acid-binding site and competitive kinetics against ATP with a Ki of 6 X 10(-6) M.

Sign in / Sign up

Export Citation Format

Share Document