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S G Rhee

Publications and source records attributed to S G Rhee.

At least 217 records · Page 12Linked to original sources
PubMed1985

The role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in Escherichia coli.

The regulation of GS activity involves two nucleotidylation cycles, the uridylylation cycle of PII and the adenylylation cycle of GS, which are catalyzed by two converter enzymes, uridylyltransferase and adenylyltransferase, respectively. The converter enzymes sense the fluctuation in the availability of nitrogen and accordingly regulate the activity of GS. On the other hand, the posttranslational modification of GS is tightly coupled to the transcriptional regulation of the glnA gene by unmodified PII protein acting as a repressor in the GS synthesis. Therefore, metabolic signals perceived by uridylyltransferase are transmitted through PII to two different levels of the regulation, namely, the posttranslational level and transcriptional level. In order to study the converter enzymes which exist in extremely low concentration, the glnD and glnE genes were cloned into a plasmid vector carrying the strong, regulatable lambda phage promoter. In this way, uridylyltransferase and adenylyltransferase were overproduced to the levels approaching 800- and 500-fold, respectively. The recombinant DNA technology also enabled us to examine the transcriptional regulation of the glnD and glnE genes. The expression of these genes was slightly repressed under nitrogen-excess conditions and the repressions were more pronounced under excess nitrogen plus carbon-limiting conditions. It was found that variations of the concentration of uridylyltransferase and adenylyltransferase also affect the rate of GS synthesis. Studies with strains harboring a multicopy plasmid, pglnD or pglnE, indicate that the elevated synthesis of the converter enzymes causes the enhancement of GS synthesis. In addition, the absence of one of the converter enzymes reduces the expression of the glnA gene. The parallel relationship between the converter enzymes and GS seems to derive from the binding capacity of the converter enzymes for the unbound PII, which is a repressor for the glnA gene. Therefore, it is believed that the metabolic regulation of the glnD and glnE genes is ultimately linked to the expression of the glnA operon.

Escherichia coli↗
PubMed1984

Conformation-specific monoclonal antibodies to glutamine synthetase in Escherichia coli.

Glutamine synthetase from Escherichia coli is composed of 12 identical subunits and exists in various forms: unadenylylated, adenylylated, divalent cation bound (taut), and divalent cation free (relaxed). The relaxed dodecamer readily dissociates into monomers upon exposure to 1 M urea or pH 8.0. Glutamine synthetase can be inactivated irreversibly by oxidizing a particular histidine residue or by incubating with methionine sulfoximine and ATP. In order to establish hybridoma monoclones that produce antibodies capable of differentiating between different conformers of glutamine synthetase, homogeneous antibodies produced from 7 clones (10-76-1, 48-76-1, 68-2-1, 57-142-2, 72-104-1, 68-3-2, 57-8-1) were characterized for their binding specificity and effects on glutamine synthetase activity. Two antibodies (10-76-1, 48-76-1) bind only to the monomeric form, two antibodies (57-142-2, 68-3-2) bind only to the dodecameric forms (taut or relaxed) and the three others (68-2-1, 72-104-1, 57-8-1) bind to both forms. At a low antibody concentration, 68-3-2 binds preferentially to taut glutamine synthetase over oxidized glutamine synthetase. None of the 7 antibodies differentiates between unadenylylated and adenylylated form. Nevertheless, the gamma-glutamyltransferase activities of the resulting antibody-glutamine synthetase complexes were influenced variably depending upon the state of adenylylation and the divalent cation.

Antibodies, Monoclonal↗
PubMed1984

Separation of glutamine synthetase species with different states of adenylylation by chromatography on monoclonal anti-AMP antibody affinity columns.

Glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli is composed of 12 identical subunits arranged in two superimposed hexagonal arrays. Therefore, partially adenylylated GS is a mixture of hybrid molecules containing different numbers (from 0 to 12) and distributions of adenylylated subunits. In an effort to separate these GS species into uniquely adenylylated molecular species, immunoadsorbants were prepared by covalent attachment of monoclonal antibodies, 72-76-1 (IgG2a and 37-2-1 (IgM), to Affi-Gel 10. The bound GS was eluted with AMP gradients. The results indicate that the IgG column and IgM column yield significantly different elution profiles; the column prepared with the IgG monoclonal antibody better resolves GS containing fewer than 3 adenylylated subunits per dodecamer, whereas the column prepared from the IgM monoclonal antibody better resolves the GS containing higher numbers (from 4 to 12) of adenylylated subunits. In addition, the results indicate that both the distribution and the number of adenylylated subunits are important factors in this separation.

Adenosine Monophosphate↗
PubMed1983

Preliminary X-ray crystallographic studies and molecular symmetry of the PII regulatory protein from Escherichia coli.

The PII regulatory protein (tetramer, Mr = 44,000) plays an important role in the regulation of glutamine synthetase in enteric bacteria. The unmodified form (PIIA) of the PII protein from Escherichia coli has been crystallized in the cubic space group I23. The unit cell dimension is a = 88.8 (+/-0.1) A, with six molecules in the unit cell (i.e. 1 subunit/asymmetric unit). The molecule has symmetry 222.

Bacterial Proteins↗
PubMed1983

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme.

Uridylyltransferase, a component of the covalent modification cascade system that controls glutamine synthetase activity in Escherichia coli, has been purified to apparent homogeneity. The purification was facilitated by the use of an E. coli strain which carries multiple copies of a ColE1-hybrid plasmid containing the glnD gene that encodes uridylyltransferase and which overproduces its synthesis by 25-fold. Gel electrophoresis and high pressure liquid chromatography studies show that the native enzyme is a single polypeptide chain of Mr = 95,000 +/- 5,000. The purified enzyme catalyzes the uridylylation as well as the deuridylylation of the regulatory protein PII, demonstrating that a single bifunctional enzyme is involved in the covalent interconversion of PII. Gel filtration studies indicate that the enzyme undergoes slow irreversible aggregation during most steps of purification with a concomitant loss of activity.

Amino Acids↗
PubMed1982

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli.

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.

Adenosine Monophosphate↗
PubMed1981

Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli.

Glutamine synthetase in Escherichia coli is regulated by adenylation and deadenylation reactions. The adenylation reaction converts the divalent cation requirement of the enzyme from Mg2+ to Mn2+. Previously, the catalytic action of unadenylated glutamine synthetase was elucidated by monitoring the intrinsic tryptophan fluorescence change accompanying substrate binding. However, due to the lack of changes in the tryptophan fluorescence, a similar study could not be done with the adenylated enzyme. In this study, therefore, an extrinsic fluor is introduced into the adenylated glutamine synthetase by adenylating the enzyme with 2-aza-1,N6-ethenoadenosine triphosphate, a fluorescent analog of ATP. The modified enzyme (aza-epsilon-glutamine synthetase) exhibits catalytic and kinetic properties similar to those of the naturally adenylated enzyme. The results of fluorometric studies on this aza-epsilon-glutamine synthetase indicated that L-glutamate and ATP bind to both Mn2+ and Mg2+ forms of the enzyme in a random order, but only the Mn2+ form is capable of forming a highly reactive enzyme-bound intermediate which is a prerequisite for the reaction with NH4+ to form products. The extrinsic fluorescence changes are also used to determine the binding constants of various substrates and inhibitors of both the biosynthetic and gamma-glutamyl transfer reactions.

Adenosine Triphosphate↗
PubMed1981

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.

Adenosine Diphosphate↗
PubMed1980

Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties.

Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C. Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection. Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII. A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g. proteolysis, phosphorylation, or adenylylation. Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases. Both enzymes are composed of 12 subunits, each approximately 51,000 daltons. However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II). Cd(II) activates EII more than EI, and only EI shows activity with Co(II). For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2. Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases. Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp. EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent. The divalent metal ion that is present can affect both specificity for analogs and substrate Km values. Kinetic binding plots (v versus [S]) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed. ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp. enzymes. For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.

Amino Acids↗
PubMed1980

Subunit interaction of adenylylated glutamine synthetase.

The glutamine synthetase (GS) activity in Escherichia coli is regulated by a bicyclic interconvertible enzyme cascade which involves the cyclic adenylylation (inactivation) and deadenylylation (activation) of GS on the one hand, and the modulation of these processes by the uridylylation and deuridylylation of Shapiro's regulatory protein on the other. The specific activity of GS in a given metabolic state is determined by the fraction of its subunits that are adenylylated, and this fraction is determined by the concentration of over 40 metabolites. Through allosteric and substrate interactions with one or more of the cascade enzymes, these metabolites alter the rates of the covalent modification and demodification reactions. By means of immunoprecipitation studies with anti-AMP specific antibodies, it has been established that the partially adenylylated glutamine synthetase, which is present in a given steady state, is a mixture of hybrid molecules containing different numbers and possibly distributions of adenylylated subunits. Partial separation of these hybrid mixtures has been achieved by affinity chromatography on Affi-Blue Sepharose columns. From immunochemical studies it is evident that anti-AMP antibodies can react with adenylylated subunits of all molecular species of GS, but that the capacities of these primary antigen-antibody reactions to yield precipitable aggregates is very dependent of the number of adenylylated subunits per molecule, and much less so upon the total concentration of adenylylated subunits present. the studies suggest that precipitability is a function either of the distribution of adenylylated subunits within hybrid species, or of the kinetics of intra- vs intermolecular bivalent interactions.

Glutamate-Ammonia Ligase↗
PubMed1980

Anti-AMP antibody precipitation of multiply adenylylated forms of glutamine synthetase from Escherichia coli: a model relating both concentration and density of antigenic sites with the antibody-antigen interaction.

Sheep antibodies directed against an AMP-bovine serum albumin conjugate exhibit highly specific binding toward AMP. These antibodies bind to the AMP moiety of adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli and to no other antigenic determinant on the protein. E coli glutamine synthetase can exist in variously modified (isomeric) forms that differ with respect to the number (0-12) and the distribution of identically adenylylated subunits [Ciardi, J. E., Cimino, F. & Stadtman, E. R. (1973) biochemistry 12, 4321-4330]. Using this enzyme, together with the AMP-specific antibodies, we have investigated the effects of the total concentration, population density, and topographical distribution of multiple identical antigenic determinants on the antigen-antibody interaction. Stopped flow fluorescence measurements show that the rate and extent of initial binding of the antibodies to the antigen are a function of the total concentration of AMP groups and are independent of the number of AMP groups der dodecamer. However, the rate of lattice formation increases with increasing epitope density, and the maximal amount of glutamine synthetase precipitated is directly proportional to the average number of adenylylated subunits per dodecamer. The data suggest that partially adenylylated enzyme preparations are composed of subpopulations of glutamine synthetase molecules that differ in their tendency to form precipitable aggregates, due presumably to differences in the topographical distribution of antigenic determinants on the surface of the enzyme. The enzyme species that form soluble immune complexes do so possibly due to intramolecular crosslinkage of the bivalent antibodies with adenylylated subunits to the exclusion of intermolecular crosslinkage.

Adenosine Monophosphate↗