Use of AMP specific antibodies to differentiate between adenylylated and unadenylylated E. coli glutamine synthetase.

Anti-AMP specific antibodies were purified by affinity chromatography of serum from sheep immunized with adenylylated bovine serum albumin. Results of immunotitration experiments and light scattering measurements show that these antibodies can be used to separate adenylylated from unadenylylated forms of $\text{E.}\text{coli}$ glutamine and to detect variations in protein configurations elicited by partial adenylylation of the enzyme or by allosteric interactions with divalent cations. These results suggest that the reaction of anti-AMP antibodies with variously adenylylated forms of glutamine synthetase can be used to investigate the dependence of immunoprecipitability on the density, absolute numbers, and possibly, the spatial distribution of multiple identical antigenic sites on a given macromolecule.     

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme.

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1). At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1). From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex. In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).     

Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Phenylalanyl-tRNA synthetase from E. coli: synergistic coupling between the sites for binding of L-phenylalanine and ATP

Binding of small ligands to the sites binding L-phenylalanine and ATP was measured by fluorescence titration technique. It is found that complex formation is not independent under conditions when both types of ligands are present in solution. The coupling is expressed as a synergistic binding rendering higher stabilities for complexes with ligand couples than expected on basis of separate binding of each ligand. In contrast, the substrate couple L-phenylalanine — ATP does not exhibit synergistic binding.     

A novel reaction catalyzed by unadenylylated glutamine synthetase from Escherichia coli. AMP-dependent synthesis of pyrophosphate and L-Glutamate from orthophosphate and L-glutamine

The unadenylylated, manganese form of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2 from Escherichia coli catalyzes a novel, AMP-dependent (reversible) synthesis of pyrophosphate and L-glutamate from orthophosphate and L-glutamine: Formula (See Text). The hydrolysis of the L-glutamine amide bond is coupled to the stoichiometric synthesis of pyrophosphate, although as PPi accumulates, additional hydrolysis of L-glutamine occurs in a secondary reaction catalyzed by the [manganese x enzyme x AMP x PPi] complex. The synthesis of PPi probably occurs at the subunit catalytic site in the positions normally occupied by the beta, gamma-phosphates of ATP. To promote PPi synthesis, AMP apparently binds to the subunit catalytic site rather than to the allosteric inhibitor site; equilibrium binding results suggest that Pi directs the binding of AMP to the active site. In this reaction, Mg2+ will not substitute for Mn2+, and adenylylated glutamine synthetase is inactive. Pyrophosphate is synthesized by the unadenylylated, manganese enzyme at approximately 2% of the rate of that of ATP in the reverse biosynthetic reaction. If P1 is replaced by arsenate, the enzymatic rate of the AMP-supported hydrolysis of L-glutamine is 100-fold faster than is PPi synthesis and is one-half the rate of the ADP-supported, irreversible arsenolysis of L-glutamine. This latter activity also is supported by GMP and IMP, suggesting that the catalytic site of glutamine synthetase has a rather broad specificity for the nucleotide base. The reactions supported by AMP directly relate to the mechanism of glutamine synthetase catalysis.     

Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.     

EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W). II. Intermediate-affinity binding sites.

The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.     

The palmityl binding sites of fatty acid synthetase from yeast.

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl-CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long-chain acyl residue both in oxygen-ester and thio-ester linkage. The lipophilic palmityl peptides were purified by column and thin-layer chromatography using organic lolvent systems. Peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4'-phosphopant-etheine-containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed.     

Limited proteolysis of glutamine synthetase is inhibited by glutamate and by feedback inhibitors.

Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.