title: Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas.
abstract: The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D014364:Tryptophan / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism*
chemical_list: D014364:Tryptophan; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase