Analytical and preparative separation of the diastereomers of L-buthionine (SR)-sulfoximine, a potent inhibitor of glutathione biosynthesis.

Buthionine sulfoximine inhibits gamma-glutamylcysteine synthetase, the enzyme catalyzing the first reaction of glutathione (GSH) biosynthesis. GSH synthesis is blocked in animals or cultured cells exposed to buthionine sulfoximine, and GSH is substantially depleted in cells or tissues with moderate to high rates of GSH utilization. Studies reported to date have used DL-buthionine (SR)-sulfoximine or L-buthionine (SR)-sulfoximine, mixtures of four and two isomers, respectively. The present report describes a chiral solvent HPLC procedure for the analytical separation of the diastereomers of L-buthionine (SR)-sulfoximine and the separation of those isomers from the unresolved diastereomers of D-buthionine (SR)-sulfoximine. L-buthionine (R)-sulfoximine was isolated preparatively by repeated crystallization of L-buthionine (SR)-sulfoximine from water; L-buthionine (S)-sulfoximine was obtained by crystallization as the trifluoroacetate salt in ethanol/hexane mixtures. The absolute configuration, bond lengths and angles of L-buthionine (R)-sulfoximine were determined by X-ray diffraction. In vitro studies demonstrate that L-buthionine (R)-sulfoximine is a relatively weak inhibitor of rat kidney gamma-glutamylcysteine synthetase; binding is competitive with L-glutamate. L-buthionine (S)-sulfoximine is a tight-binding, mechanism-based inhibitor of the enzyme. Since L-buthionine sulfoximine is initially bound as a transition-state analogue, identification of the inhibitory diastereomer elucidates the steric relationships among ATP, glutamate, and cysteine within the active site. When administered to mice, L-buthionine (S)-sulfoximine (0.2 mmol/kg) was as effective as L-buthionine (SR)-sulfoximine (0.4 mmol/kg) in causing GSH depletion in liver, kidney, and pancreas. L-Buthionine (R)-sulfoximine (0.2 mmol/kg) did not cause significant GSH depletion in liver or pancreas. The L-(R)-diastereomer caused a modest GSH depletion in kidney that is tentatively attributed to interference with gamma-glutamylcyst(e)ine transport.     

Characterization of Salmonella typhimurium Strains Sensitive and Resistant to Methionine Sulfoximine

Methionine sulfoximine inhibits the growth of Salmonella typhimurium at a concentration of 50 muM, and the addition of glutamine, but not glutamate, is sufficient to overcome this inhibition. The analogue causes 50% inhibition of glutamine synthetase activity at 2 to 4 muM and of glutamate synthase at 2 to 3 mM when these enzymes are assayed in vitro. No inhibition of glutamate dehydrogenase activity is observed at analogue concentrations as high as 50 mM. Two mutants selected for their resistance to methionine sulfoximine inhibition have a partial growth requirement for glutamine and a reduction in the glutamine synthetase and glutamate synthase activities. The sensitivity of the remaining glutamine synthetase activity in these mutants to methionine sulfoximine inhibition appears unaltered, and the lesions conferring the analogue resistance may not affect glutamine synthetase directly.     

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.     

Mechanism of inhibition of pea chloroplast glutamine synthetase by methionine sulfoximine

In the presence of ATP and Mg2+ L-methionine sulfoximine irreversibly inhibits homogeneous glutamine synthetase (EC 6.3.1.2) from pea chloroplasts (I0.5 = 1.0 x 10(-7) M; Ki = 6.25 . 10(-8) M. Glutamate (but not NH4Cl) exerts a protective effect, which is enhanced when glutamate and NH4Cl are simultaneously present in the reaction mixture. The inhibiting action of L-methionine sulfoximine with respect to glutamate is of a mixed type. ATP and Mg-ATP produce the same non-competitive protective effect on L-methionine sulfoximine. The data obtained suggest that the formation of a quaternary complex (or a transition state) between the enzyme and all its substrates is essential for the catalysis.     

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.     

Subunit interaction elicited by partial inactivation with L-methionine sulfoximine and ATP differently affects the biosynthetic and gamma-glutamyltransferase reactions catalyzed by yeast glutamine synthetase

Yeast glutamine synthetase can be irreversibly inactivated in the presence of L-methionine sulfoximine, ATP, and a divalent cation Mn2+ or Mg2+. Kinetic studies with partially inactivated enzymes show that inactivation of a given subunit in the octameric glutamine synthetase affects the activities of its neighboring subunit such that the rate of the inactivation as well as the gamma-glutamyltransferase activity of the noninactivated subunits decreases while their biosynthetic activity is enhanced. This outcome of subunit interaction is the same irrespective of whether Mn2+ or Mg2+ is used to fulfill the divalent cation requirement of glutamine synthetase for the inactivation reaction and the gamma-glutamyltransferase reaction. Although only Vmax is affected in the gamma-glutamyltransferase assay, both Km (glutamate) and Vmax are changed in the biosynthetic assay.     

Differential inhibition of glutamine and gamma-glutamylcysteine synthetases by alpha-alkyl analogs of methionine sulfoximine that induce convulsions.

The alpha-methyl and alpha-ethyl analogs of methionine sulfoximine, like methionine sulfoximine, induce convulsions in mice and inhibit glutamine synthetase irreversibly; alpha-ethylmethionine sulfoximine is approximately 50% as inhibitory as methionine sulfoximine and alpha-methylmethionine sulfoximine. However, whereas alpha-methylmethionine sulfoximine and methionine sulfoximine inhibit gamma-glutamylcysteine synthetase markedly, alpha-ethylmethionine sulfoximine does not, nor does administration of the alpha-ethyl analog produce the decrease in tissue glutathione levels found after giving methionine sulfoximine or its alpha-methyl analog. The findings strongly indicate that methionine sulfoximine-induced convulsions are closely associated with inhibition of glutamine synthetase rather than with inhibition of gamma-glutamylcysteine synthetase. The alpha-alkyl methionine sulfoximine analogs cannot be catabolized via the corresponding alpha-keto or alpha-imino acids, and, like other alpha-substituted amino acids, are probably not metabolized to a significant extent in vivo; this suggests that the amino acid sulfoximine molecules themselves, rather than their metabolites, are directly involved in the induction of convulsions. Possible explanations for the reported lack of correlation between the occurrence of convulsions and the levels of glutamine synthetase activity (and its substrates and product) are considered. The findings suggest that studies on the mechanism of induction of convulsions may be extended significantly and refined in biochemical terms by the use of other structurally modified convulsant molecules.     

Glutamine synthetase from Salmonella typhimurium: manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme.

Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K_D values of 3.7 × 10^?6 and 1.7 × 10^?4m, while at pH 6.8 these values were 1.1 × 10^?5 and 1.0 × 10^?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a K_D value of 1.0 × 10^?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (K_D = 3.7 × 10^?6) are de-enhanced (?_b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?_b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.     

Kinetic,ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells

Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50–57 M, of which 30–40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (±0.2) M, very near K_d for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound⁵⁴Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. -Methyl-d,l-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme -glutamyl-cysteine synthetase prior to specific inactivation of GS by -ethyl-d,l-methionine-S,R-sulfoximine. Even inthe presence of 2–3 mM Mg(II), with only 5–10 M Mn(II) present, approximately 20–30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.     

Recognition of a cysteine substrate by E. coli gamma-glutamylcysteine synthetase probed by sulfoximine-based transition-state analogue inhibitors

A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coli gamma-glutamylcysteine synthetase (gamma-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution. The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.     

Uridylylation of the P(II) protein in the photosynthetic bacterium Rhodospirillum rubrum.

The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.     

Chromium(III)-adenosine triphosphate as a paramagnetic probe to determine intersubstrate distances on pyruvate kinase. Detection of an active enzyme-metal-ATP-metal complex.

The interaction of CrATP, a stable, substitution-inert, paramagnetic tridentate complex of ATP, with muscle pyruvate kinase has been studied by measuring the effects of CrATP on the kinetics of pyruvate enolization and on the longitudinal nuclear magnetic relaxation rate (1/T1) of the protons of water and the protons and carbon atoms of pyruvate to investigate the existence and activity of bimetallic enzyme-M(II)-CrATP complexes and to determine intersubstrate distances on a kinase. The paramagnetic effect of CrATP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP yielded characteristic enhancements of 1/T1 for the binary enzyme-CrATP, ternary enzyme-Mg(II)-crATP, and quaternary enzyme-Mg(II)-crATP-pyruvate complexes of 3.5, 1.7, and 1.2 and dissociation constants of CrATP of 400, 200, and 200 muM, respectively. From the frequency dependence of 1/T1, the number of fast exchanging water protons in the coordination spheres of Cr(III) is approximately 6 in CrATP and in both the ternary enzyme-Mg(II)-CrATP complex and the quaternary enzyme-Mg(II)-CrATP-pyruvate complex. The paramagnetic effect of enzyme-bound Mn(II) on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn(II) complex with CrATP decreases the characteristic enhancement due to Mn(II) from 24 +/- 3 to 6 +/- 1. Titration of the ternary eznyme-Mn(II)-pyruvate complex with CrATP decreases the enhancement from 6 +/- 1 to 0.5 +/- 0.1. The affinity of the enzyme for Mn(II) is increased 2-fold upon binding of CrATP as indicated by decreases in the amplitude of the EPR spectrum of free Mn(II). The dissociation constants of CrATP from the enzyme-Mn(II)-CrATP complex, the enzyme-CrATP-pyruvate complex, and the enzyme-Mn(II)-CrATP-pyruvate complex are all 200 muM. The observed titration behavior, the characteristic enhancement values, the tightening by Mg(II) of the binding of CrATP to the enzyme, and the tightening of the binding of Mn(II) to the enzyme by CrATP establish the existence of enzyme-M(II)-CrATP and enzyme-M(II)-CrATP-pyruvate complexes containing two cations, Mg(II) or Mn(II) and Cr(III), at the active site.     

Identification of nonprotein ligands to the metal ions bound to glutamine synthetase

Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.     

Magnetic resonance studies of Mn(II)-, Mn(III)-, and Fe(III)-conalbumin complexes.

Apoconalbumin binds Mn(II) at two sites with association constants of K1 = 7 (+/- 1) X 10(4) and K2 = 0.4 (+/- 0.25) X 10(4) M-1. The binding is tighter in the presence of excess bicarbonate resulting in K1 = 1.8 (+/- 0.2) X 10(5) and K2 = 3 (+/- 2) X 10(4) M-1. The electron paramagnetic resonance spectrum (at both 9 and 35 GHz) of Mn(II) bound at the tight site reveals a rhombic distortion (lambda = E/D approximately equal to 0.25-0.31) in the protein ligand environment of the mental ion. An evaluation of the 1/pT1p, paramagnetic contribution to the longitudinal relaxation rate of solvent protons with Mn(II)-, Mn(III)-, and Fe(III)-derivatives of conalbumin revealed that the mental ion in each site of conalbumin is accessible to one water molecule. For Mn(II)-conalbumin and Mn(III)-conalbumin species, inner coordination sphere protons are rapidly exchanging with the bulk solvent, while slow exchange conditions prevail for Fe(III)-conalbumin.     

Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae

Ammonium suppresses nitrogenase activity in Anabaena flos-aquae (Lyng) Breb. at all pH values tested. l-Methionine-dl-sulfoximine at 1 millimolar totally inhibited glutamine synthetase, and 10 micromolar partially inhibited. Both concentrations protected nitrogenase activity from ammonium-induced suppression at pH 7.1 and 8.1. At pH 9.3 and 10.2, methionine sulfoximine did not alleviate the suppression of nitrogenase by ammonium. This pH-dependent protection of nitrogenase activity is a result of the noncompetitive inhibition of the ammonium transporter by methionine sulfoximine. At pH 7.1 and 8.2, ammonium is protonated and methionine sulfoximine inhibits its entry into the cell. At pH 9.3 and 10.2, unprotonated ammonia is abundant and may enter the cell independent of the transport system. The effects of ammonium are closely mimicked by the ammonium analog methylamine. These results suggest that ammonium per se is an important in vivo regulator of nitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re-evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter.     

Methionine sulfoximine and phosphinothricin--glutamine synthetase inhibitors and activators and their herbicidal activity (A review)

Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairments of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in a concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.     

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.     

The Effect of Phosphinothricin (Glufosinate) on Glutathione Synthesis in Plants

The inhibitory effect of DL-phosphinothricin (glufosinate) on glutathione synthesis was studied in vivo and in vitro. The influence of phosphinothricin on γ-glutamylcysteine synthetase was compared with the already known effects of l -buthionine sulfoximine and l -methionine sulfoximine. The results showed that phosphinothricin and buthionine sulfoximine are inhibitors of γ-glutamylcysteine synthetase of plants. With both substances the enzyme was inhibited by 50 % at a concentration of 7 . 10^?4M (pI₅₀ = 3.15). Methionine sulfoximine reduced the enzyme activity by 50% at 5 . 10^?2 M (pI₅₀ = 1.30). It is discussed that the target enzyme of phosphinothricin is the glutamine synthetase whereas the γ-glutamylcysteine synthetase is only an accessory target.