Affinity labeling of phenylalanyl-tRNA synthetase from E.coli MRE-600 by E.coli tRNAphe containing photoreactive group

Abstract

The photoinduced reaction of phenylalanyl-tRNA synthetase (B.C. 6.1.1.20) from E.coli MRE-600 with tRNAAPhe containing photoreative p-N3-C6H4-NJHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAAPhe) was investigated. inhibitio of the photoinduced reaction. Therefore, the reaction of [14c -Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNAenzyme interaction.

Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

“…The unique chemical reactivity of the 4-thiouridine residue present at position 8 in many E. coli tRNAs has previously allowed preparation of a variety of site-specific fluorescent, spin-labeled, and photoreactive tRNA derivatives (Hara & Horiuchi, 1970; Yang & Soil, 1973;Budker et al, 1974; Caron & Dugas, 1976; Gorshkova et al, 1976;Wetzel & Soil, 1977;Johnson et al, 1977;Ofengand et al, 1977;Pande & Wishnia, 1985). In the present work, we have developed a procedure suitable for coupling a lysine-reactive cross-linker to 4-thiouridine residues.…”

“…Photoaffinity probes have previously been coupled to 4thiouridine residues in several E. coli tRNAs, and covalent cross-linking to cognate synthetases has been observed (Budker et al, 1974;Gorshkova et al, 1976; Wetzel & Soil, 1977); however, no peptide sequences have emerged from these studies. In our experience, higher yields of specific peptides are obtained by using affinity-labeling derivatives directed to react with specific amino acid residues in the protein.…”

Equations of the kinetic curves of affinity labelling of biopolymers with the reagents consumed in parallel reactions in solution

“…Kinetics of affinity labelling is used to estimate the affinity of the respective reagents to specific proteins which protect them against modification and inactivation. Kinetics of the labelling of multisubstrate enzymes with one substrate analog in the presence of other substrates was demonstrated to reflect the interactions between the sites of these enzymes [3,4].…”

Affinity labelling of yeast hexokinase with benzylamide derivatives of adenosine mono‐ and triphosphates bearing an alkylating group

“…The affinity labelling kinetics were expected in some cases to be highly sensitive to the local change of conformation of a biopolymer in the vicinity of the reactive group of the label within the specific complex [4]. Therefore, we have studied the reaction of PI isoenzyme of yeast hexokinase with two alkylating reagents: 4-(N-2-chloroethyl-N-methylamino)-benzylamides of ATP; and AMP (C1RCH2NHpppA and C1RCH2NHpA) in the conditions favouring either the monomeric or dimeric form of hexokinase.…”