Co-operative effects in affinity labeling reveal the interaction of tRNA-recognition centers of phenylalanyl-tRNA synthetase.
A mathematical treatment of affinity labeling of the enzymes is presented. The model considered involves a dimeric enzyme with identical ligand binding sites. Equations are derived which describe the kinetics of modification; mutual influence of ligand molecules on association, on the rate of covalent attachment and the possibility of the existence of different sites of modification are taken into account. Experimental data on affinity labeling of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) of Escherichia coli MRE-600 with N-bromoacetyl-[14C]phenylalanyl-tRNA are treated in terms of the model suggested. The affinity (association constant value) of the tRNAPhe analog molecule towards the enzyme is only slightly affected by another molecule, whereas the reaction rate constant of covalent attachment decreases significantly. The latter is assumed to be due to acceptor site change in the complex containing two molecules of the tRNAPhe analog.
D000345:Affinity Labels / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D000345:Affinity Labels; D012346:RNA, Transfer, Amino Acyl; C032617:tRNA, N-bromoacetylphenylalanine-; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase