Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.     

High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-beta-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be deductible on the basis of complementation testing.     

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas

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.     

Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator

Three spontaneous derivatives of Streptococcus faecium ATCC 9790, originally isolated as conditionally Triton X-100 detergent-resistant at 25 degrees C, displayed normal penicillin-induced rates of lysis at 37 degrees C but substantially reduced rates of lysis and killing at 25 degrees C. The addition of exogenous unsaturated fatty acids at 25 degrees C restored wild-type penicillin lysis rates.     

S-Adenosylmethionine synthetase from Escherichia coli

Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.     

Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton.

Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain. Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme. These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence. Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex. This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero. It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J. P. (1975), J. Mol. Biol. 94, 1.).     

Autoadenylation of tryptophanyl-tRNA synthetase

Incubation of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) deprived of endogenous tryptophan, with [14C]ATP and without [gamma-32P]ATP, causes an appearance of radioactivity in protein due to formation of adenylated enzyme, [14C]AMP-E. Examination of the properties of the [14C]AMP-E thus obtained led to the conclusion that AMP is linked to the protein molecule via a macroergetic phosphoanhydride bond. ATP is formed when AMP-E is incubated with PPi. However, no tryptophanyl adenylate formation was observed when AMP-E was treated with tryptophan. The functional role of AMP-E remains obscure.     

Crystallization of succinyl-CoA synthetase from Escherichia coli

Well formed, tetragonal prisms of succinyl-CoA synthetase from Escherichia coli have been crystallized at room temperature from ammonium sulfate and mixtures of sodium and potassium phosphates. A systematic survey of the conditions for crystallization of the enzyme has been carried out. This has shown the addition of a small amount of an organic solvent (acetone, 2-methyl-2,4-pentanediol, tert-butyl alcohol, or tertamyl alcohol) to the phosphate media and of CoA to the sulfate media to be beneficial in producing large, single crystals suitable for analysis by x-ray diffraction methods. Preliminary examination of precession photographs reveals that the crystals from phosphate media have a unit cell of symmetry P4222 with dimensions a = b = 94 A and c = 248 A. Evidence suggests that there may be only half of the (alpha beta)2 tetramer/asymmetric unit in these crystals. The crystals from ammonium sulfate media have unit cell dimensions of a = b = 99 A and c = 399 A, a space group of P4122 (P4322), and one tetramer/asymmetric unit. They diffract to a resolution of 3.4 A. Both crystal types have large solvent contents of about 65% of the unit cell volumes. A parameter called "quality index" is introduced to facilitate comparison of crystals grown under a variety of conditions with respect to their quality of x-ray diffraction.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.