Anticooperative Binding of l‐Tryptophan to Tryptophanyl‐tRNA Synthetase from Beef Pancreas

Abstract

Equilibrium dialysis and gel filtration studies show that tryptophanyl-tRNA synthetase from beef pancreas binds two molecules of L-tryptophan per dimer in an anticooperative way.The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein-tryptophan system upon formation of the complex is d c 2 9 2 = 10400 i 1000 M-' cm-' per dimer.Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 C are respectively K I = 2.0 ? 0.5 pM and KZ = 10 -t 4 pM. They are respectively K 1 = 1 -+ 0.25 p M and Kz = 20 i 8 pM if one considers a sequenced binding of the two tryptophan molecules.The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.Tryptophanyl-tRNA synthetase belongs to the dimeric x2 class of aminoacyl-tRNA synthetases whether it comes from procaryotes such as Escherichiu coli [ 1 ] or BuciIIus strurotl?ermophilus [2] or from eucaryotes such as yeast [ 3 ] , beef [4,5] or man [6]. For such enzymes two questions arise. (a) Are the two subunits functionally equivalent or not? It has been suggested, for example, in the case of the tyrosine enzyme of B. strurother-mo~~/iilu.~, which shows nonequivalence in the binding sites [7], that this nonequivalence plays a role in the catalytic mechanism [S]. (b) Is the binding process accompanied by a change in the protein conformation? This is possibly the case a s soon as the binding is not Michaelian. High binding energy is usually observed in the formation of the specific synthetase-substrate complexes. at least as far as the amino acid and the tRNA are concerned [9]. This high binding energy, which is part of the specificity process, should mainly concern ~~

Kinetic Anticooperativity in Pre‐Steady‐State Formation of Tryptophanyl Adenylate by Tryptophanyl‐tRNA Synthetase from Beef Pancreas

“…The tryptophan dissociation constants for the high-affinity and the low-affinity sites found by binding experiments in the absence of ATP [15] are not very different from the apparent dissociation constants derived under pre-steady state conditions by stopped-flow experiments in the presence of ATP-Mg (this work). The presence of ATP-Mg changes the apparent binding of tryptophan by a factor of less than two and similarly the presence of tryptophan changes the apparent binding of ATP-Mg by a factor of less than two.…”

“…The tyrosine enzyme from E. coli has two binding sites but only one of them is able to make tyrosyl adenylate [I 31 ; on the contrary that of B. stearo-thermophilus is able to make two adenylates per dimer [13]. The tryptophan enzyme from E. coli has two equivalent binding sites for tryptophan [14] while that of beef pancreas has two anticooperative sites for the amino acid [15]. For this latter protein a covalent tryptophanyl-enzyme derivative has been described, in which a maximum of one amino acid is bound to the enzyme [16].…”

Kinetic evidence for half-of-the-sites reactivity in tRNATrp aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

“…This work describes an attempt to use these possibilities in the case of beef tryptophanyl-tRNA synthetase, an a2 dimeric enzyme with two sets of binding sites for its substrates (Dorizzi et al, 1977;Zinoviev et al, 1977;Graves et al, 1979Graves et al, , 1980.…”

Towards an Integrative Understanding of tRNA Aminoacylation–Diet–Host–Gut Microbiome Interactions in Neurodegeneration

“…The different affinities of the two subunits of bovine pancreatic TrpRS for tryptophan were observed by Graves et al [ 129 ] and Mazat et al [ 130 ]. Taking an initial symmetrical dimeric TrpRS the two dissociation constants for tryptophan at pH 8 and 25 °C are respectively K 1 = 2.0 ± 0.5 µM and K 2 = 10 ± 4 µM [ 129 ]. They are respectively K 1 = 1 ± 0.25 µM and K 2 = 20 ± 8 µM if one considers a sequenced binding of the two tryptophan molecules.…”

“…Dissociation constant (Kdis) for tryptamine in the tryptophan-dependent ATP-[32P] pyrophosphate exchange catalyzed by bovine pancreatic TrpRS after covalent binding of 1 mole of R-Trp-tRNA to the enzyme (one site of modified TrpRS was occupied after the binding) is 13 ± 4 × 10 −7 M versus tryptamine Kdis 3.6 ± 0.6 × 10 −7 M for the native enzyme [ 128 ]. The different affinities of the two subunits of bovine pancreatic TrpRS for tryptophan were observed by Graves et al [ 129 ] and Mazat et al [ 130 ]. Taking an initial symmetrical dimeric TrpRS the two dissociation constants for tryptophan at pH 8 and 25 °C are respectively K 1 = 2.0 ± 0.5 µM and K 2 = 10 ± 4 µM [ 129 ].…”