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.

Pyrophosphate‐caused inhibition of the aminoacylation of tRNA by the leucyl‐tRNA synthetase from Neurospora crassa

“…The value K4 x k + 3 / k -= 120 pM. If the value of k + , / k -, for the leucyl-tRNA synthetase is of the same order of magnitude as reported by Merle et al [13] for the tryptophanyl-tRNA synthetase from beefpancreas: k + 3 / k -3 = 42 sP1/15O s -l = 0.28, the value of K4 would be 430 pM. Therefore high-affinity binding of pyrophosphate need not be assumed to explain the low K:"' values from the measurements.…”

Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

“…With F5, C38, M129 and H43 replacing the four hydrogen‐bonding residues in human TrpRS, the tryptophan‐binding pocket appears to be more hydrophobic in B. stearothermophilus TrpRS (Figure 5C). Given that tryptophan binds with a similar affinity to eukaryotic and prokaryotic TrpRSs (Merle et al , 1984; Jia et al , 2002; Ewalt et al , 2005), hydrophobic interactions may play a more important role in B. stearothermophilus TrpRS than in human TrpRS, in order to compensate for weaker hydrogen bonding interactions in B. stearothermophilus TrpRS. The difference in mode of tryptophan binding by the two orthologs of TrpRS provides a structural rationale for designing antibiotics that specifically inhibit prokaryotic TrpRS.…”

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNATrp aminoacylation

“…Under these conditions, the complexes were synthesized within a few minutes (Mazat et al, 1982). Due to the anticooperative binding of tryptophan to the enzyme, Kix = 1.6 and Ki2 = 18.5 µ (Merle et al, 1984), the synthesis of tryptophanyl adenylate led to 74% of monoand to 13% of bis(tryptophanyl adenylate)-enzyme complexes when enzyme and tryptophan were in stoichiometric amounts (0.4 µ each).…”

“…The limiting event in a complex enzyme reaction is usually expected to be one of the chemical steps on the reaction pathway. In the case of the aminoacylation of tRNATrp by the dimeric tryptophanyl-tRNA synthetase from beef, the activation of tryptophan into tryptophanyl adenylate [rate constant 40 s'1 per active site (Merle et al, 1984)], and its transfer from the adenylate to tRNATrp [rate constant 35 s'1 per dimer (Trezeguet et al, 1986)], when individually studied, do not appear to be slow enough to account for the overall steady-state rate constant of tRNATrp aminoacylation by the enzyme (6.5 s'1; Merault et al, 1978). Therefore, another step has to be sought.…”