Investigation of the mechanism of phosphinothricin inactivation of Escherichia coli glutamine synthetase using rapid quench kinetic technique.

A number of slow tight-binding inhibitors are known for glutamine synthetase that resemble the geometry of the tetrahedral intermediate formed during the enzyme-catalyzed condensation of gamma-glutamyl phosphate and ammonia. One of these inhibitors, phosphinothricin [L-2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid], has been investigated by rapid kinetic methods. Phosphinothricin not only exhibits the kinetic properties of a slow tight-binding inhibitor but also undergoes phosphorylation during the course of the ATP-dependent inactivation. The acid lability of phosphinothricin phosphate enabled investigation of the kinetics of glutamine synthetase inactivation using rapid quench kinetic techniques. The rate-limiting step in the inhibition reaction is the binding of inhibitor (0.004-0.014 microM-1 s-1) and/or a conformational change associated with binding, which is several orders of magnitude slower than the binding of ATP. The association rate of phosphinothricin depends on which metal ion is bound to the enzyme (Mn2+ or Mg2+). With Mn2+ bound to glutamine synthetase the rate of association and the phosphorylation rate are faster than when Mg2+ is bound. The data are interpreted with use of a model in which the binding of a substrate analogue with a tetrahedral moiety enhances the phosphorylation rate of the reaction intermediate; however, the initial binding interaction is retarded because the enzyme has to bind a molecule that has a "transition-state" geometry rather than a ground-state substrate structure. During the course of the inactivation, progressively slower rates for binding and phosphoryl transfer were observed, indicating communication between active sites.     

Determination of the mechanism of the argininosuccinate synthetase reaction by static and dynamic quench experiments

The reactions catalyzed by argininosuccinate synthetase have been examined by the use of static and dynamic quench techniques. The time course of the forward reaction (22 degrees C) at pH 8.0 is characterized by a "burst" of AMP formation upon quenching with acid that is equivalent to 0.59 mol of enzyme. The pre-steady-state rate is followed by a slower steady-state rate of 0.60 s-1. The rate constant for the transient phase is 9.7 s-1. The time course for the formation of argininosuccinate is linear and shows neither a "lag" nor a burst phase. These results have been interpreted to mean that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. In the presence of aspartate the rate constant for the formation of citrulline adenylate (6.2 s-1) from ATP and citrulline is 7 times faster than the rate of formation of argininosuccinate from aspartate and citrulline adenylate (0.9 s-1). This suggests that the second step is predominantly rate limiting. The rate constant for the formation of citrulline adenylate in the absence of enzyme-bound aspartate (0.01 s-1) is 600 times slower than when aspartate is present. This indicates that the binding of aspartate to the enzyme regulates the formation of the intermediate. These results are in complete accord with our previously published steady-state kinetic scheme showing sequential addition of substrates.     

Cooperative effects of CTP on calf liver CTP synthetase.

In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.     

Synthesis of N4-substituted CTP by mammalian CTP synthetase

Highly purified CTP synthetase from Ehrlich ascites tumor cells catalyzes the formation of N4-substituted CTP from UTP and hydroxylamine and its derivatives. The products with hydroxylamine and O-methylhydroxylamine were identified as N4-hydroxyCTP and N4-methoxyCTP by absorption spectra and chromatographic behavior on Dowex column, respectively. The weak nucleophilic amines such as methylamine, ethylamine or diethylamine and a less nucleophilic amine, sulfamic acid, did not react with UTP. These results suggest that the nucleophilicity and basicity of amines are important in the enzymic reaction with UTP.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.     

Cyclopentenylcytosine triphosphate. Formation and inhibition of CTP synthetase

Cyclopentenylcytosine (CPEC) is phosphorylated in L1210 cells with CPEC triphosphate as the major metabolite. Partially purified uridine-cytidine kinase catalyzes the initial phosphorylation of cyclopentenylcytosine with an apparent Km of 196 +/- 9 microM, and cyclopentenylcytosine is a competitive inhibitor of cytidine phosphorylation by this enzyme with a Ki value of 144 +/- 14 microM. Examination of the CTP synthetase activity in extracts of L1210 cells revealed a dose-dependent decrease on exposure of cells to CPEC. Synthesis of CPEC triphosphate by an enzymatic method permitted direct examination of the inhibition of partially purified CTP synthetase. CPEC triphosphate inhibited bovine CTP synthetase with a median inhibitory concentration of 6 microM, whereas CPEC mono- and diphosphates were ineffective. CTP synthetase showed a classical Michaelis-Menten hyperbolic plot of velocity and UTP concentration in the presence of saturating concentrations of ATP and glutamine, but CPEC triphosphate induced sigmoidal kinetic plots. The Hill coefficient was calculated to be 3.2.     

The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).     

Substrate specificity of CTP synthetase from Escherichia coli

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.     

Determination of the rate-limiting steps and chemical mechanism of fructokinase by isotope exchange, isotope partitioning, and pH studies.

Isotope exchange studies show that beef liver fructokinase has a random kinetic mechanism in which release of fructose from the enzyme is slower than that catalytic reaction. The stickiness of fructose in the presence of MgATP is confirmed by isotope partition studies, which show it to be released 0.53 times as fast as V1/Et in the presence, and 80--130 times as fast in the absence of MgATP. Fructose-1-P release from it binary complex is not at all rate limiting in the forward direction since no exchange of MgADP back into MgATP could be observed during the forward reaction. Failure to find any isotope effect by the equilibrium perturbation method with [1-18O]fructose (upper limit, 1.003, shows that P--O bond cleavage or formation is not rate limiting. The pH profiles for the forward reaction show a group (probably carboxyl with pK 5.7-6.0 and deltaHion = 0) that must be ionized and a group (perhaps lysine, with pK 9--10, and deltaHion 5-9 kcal/mol) which must be protonated for activity. The profile for the back reaction shows only a group with pK 5.5--6 that must be protonated for activity. A chemical mechanism is proposed in which a carboxyl group on the enzyme accepts a proton from the 1-hydroxyl of fructose during the forward reaction and donates it back during the reverse reaction.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).     

New insights into butterfly-environment relationships using partitioning methods

Variation partitioning and hierarchical partitioning are novel statistical approaches that provide deeper understanding of the importance of different explanatory variables for biodiversity patterns than traditional regression methods. Using these methods, the variation in occupancy and abundance of the clouded apollo butterfly (Parnassius mnemosyne L.) was decomposed into independent and joint effects of larval and adult food resources, microclimate and habitat quantity. The independent effect of habitat quantity variables (habitat area and connectivity) captured the largest fraction of the variation in the clouded apollo patterns, but habitat connectivity had a major contribution only for occupancy data. The independent effects of resources and microclimate were higher on butterfly abundance than on occupancy. However, a considerable amount of variation in the butterfly patterns was accounted for by the joint effects of predictors and may thus be causally related to two or all three groups of variables. Abundance of the butterfly in the surroundings of the focal grid cell had a significant effect in all analyses, independently of the effects of other predictors. Our results encourage wider applications of partitioning methods in biodiversity studies.     

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.     

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Deltaura8Delta double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.