Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.
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The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used. (+info)
Properties of anthranilate synthetase component II from Pseudomonas putida.
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The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization. AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide. Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM. Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex. In both cases glutamine protects against alkylation and exhibits competitive kinetics. When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone. Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate. These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate. This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate. (+info)
Escherichia coli chorismate synthase: a deuterium kinetic-isotope effect under single-turnover and steady-state conditions shows that a flavin intermediate forms before the C-(6proR)-H bond is cleaved.
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We report the observation of a deuterium kinetic isotope effect for the conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (6proR2HV = 1.13 +/- 0.03) using recombinant chorismate synthase from Escherichia coli. Similar isotope effects were observed for the decay of a spectroscopically characterized flavin intermediate (6proR2Hk = 1.17 +/- 0.04) during single-turnover experiments. The main rate-limiting steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either before or concomitant with its decay. (+info)
Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 1. The effect of NaCl and its use in a new purification involving affinity chromatography on sepharosyl-phenylalanine.
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A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme. NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine. (+info)
Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 2. Evidence for identical subunits catalysing the two activities.
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On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits. It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit. (+info)
Anthranilate synthase from Bacillus subtilis. The role of a reduced subunit X in aggregate formation and amidotransferase activity.
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With respect to its sulfhydryl groups, subunit X can exist in at least two forms, oxidized (Xox) and reduced (Xre). The importance of the Xre form for the formation of an EX complex and for amidotransferase activity has been examined. Subunit Xre is rapidly inactivated by p-chloromercuribenzoate and bromopyruvate, whereas subunit Xox, which is not catalytically functional in amidotransferase activity, is not affected. The glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) has no effect on Xre alone but rapidly inactivates the EXre complex. DON-inactivated subunit X cannot be reactivated by 2-mercaptoethanol but can be readily displaced from subunit E by free subunit Xre. The integrity of the EXre complex is maintained following gel filtration on Sephadex G-100 in the presence of glutamine and 2-mercaptoethanol, thus the binding of glutamine to the complex does not require the binding of other substrates. Subunit Xox, however, does not aggregate with subunit E since no EXox complex is found following gel filtration on Sephadex G-100 in the presence of glutamine and in the absence of 2-mercaptoethanol. Thus, a reduced sulfhydryl group(s) is not only essential for amidotransferase activity but also for the formation of the aggregate as well. The following model is proposed to explain these results. Free subunit Xre does not bind DON or glutamine to the catalytically functional sulfhydryl group. Upon aggregation with subunit E, however, the glutamine or DON binds to the glutamine catalytic site on subunit Xre and amidotransfer or alkylation occurs. An EX complex which has been alkylated by DON can be readily dissociated and it is suggested that following catalysis the EX complex may also dissociate. (+info)
Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.
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The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity. (+info)
Binding of a high-energy substrate conformer in antibody catalysis.
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Enzymes can substantially increase the probability of a reaction by exploiting binding energy to preorganize their substrates into reactive conformations. Similar effects are likely to be important in a wide variety of designed catalysts, including catalytic antibodies. Transferred nuclear Overhauser effects have been used here to investigate how an antibody possessing chorismate mutase activity binds its flexible substrate molecule chorismate. The conversion of chorismate to prephenate by way of a Claisen rearrangement requires the substrate to adopt an energetically disfavored diaxial conformation in which the enolpyruvyl side chain is positioned over the six-membered ring. The antibody, which was elicited by a conformationally restricted transition state analog for this reaction, appears to bind this high-energy substrate conformer preferentially, as judged by diagnostic intramolecular transferred nuclear Overhauser effects. Inhibitor studies with the transition state analog confirm that preorganization takes place exclusively at the antibody active site. These results thus provide strong physical evidence for a direct relationship between the properties of a catalytic antibody and the structure of the transition state analog originally used to elicit the immune response. (+info)