Proline biosynthesis in Escherichia coli. Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The Km for DL-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM. The Vmax is approx. 8.0 units per mg protein. The reaction is highly specific for the DL-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of gamma-glutamyl phosphate, 3-(phosphonoacetylamido)-L-alanine, is competitive with respect to DL-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation.     

Proline biosynthesis in Escherichia coli. Stoichiometry and end-product identification of the reaction catalysed by glutamate semialdehyde dehydrogenase.

The stoichiometry of the oxidative phosphorylation of glutamic acid 5-semialdehyde by gamma-glutamyl phosphate reductase (glutamate semialdehyde dehydrogenase) has been established. Equimolar amounts of NADP+ and L-glutamic acid 5-semialdehyde are consumed and equimolar amounts of 5-oxiopyrroilidine-2-carboxylic acid and NADPH are formed. The end-product of the reaction is demonstrated to be 5-oxopyrrolidine-2-carboxylic acid, probably arising from the true end-product gamma-glutamyl phosphate.     

Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli

Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied. The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C. When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating. From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM. ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD. Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity. The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000. The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity. Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in K_m. Apparent K_m values are 6.4 μM for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 μM for NAD.     

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.     

Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme

The pattern of protein synthesis in hepatoma cell clones was analysed by two-dimensional separation of [35S]methionine-labelled proteins. The clones were derived from the differentiated Reuber H 35 hepatoma and showed differences in the expression of a number of liver-specific functions and the resistance to the growth-inhibitory effect of glucocorticoids. Five protein spots were observed in the extracts of the differentiated Faza 967 cells that were absent from the electrophoretogram of the dedifferentiated H 56 cells. This clone, on the other hand, displayed six spots absent from Faza 967 cells. The growth of both Faza 967 and H 56 cells was strongly inhibited by 1 microM dexamethasone. The dexamethasone-resistant clone 2, a dedifferentiated derivative of Faza 967 cells, synthesized two polypeptides that were not present in Faza 967 or H 56 cells and produced four polypeptides at a lower level than Faza 967 cells. The examination of the short-term effect of dexamethasone on protein synthesis in Faza 967 cells revealed nine induced and one repressed protein spots, which appeared to be in good agreement with earlier published data. It is concluded that dedifferentiation, although bringing about marked changes in certain liver-specific functions, such as enzyme activities or protein secretion, affects only a relatively small fraction of the genes expressed.     

The isozymic nature and kinetic properties of glutamate dehydrogenase from safflower seedlings

Levels of glutamate dehydrogenase (GDH) [L-glutamate: NAD oxidoreductase(deaminating), EC 1.4.1.2 [EC] ] from safflower roots and cotyledonsincreased (?2.7) and decreased ( ?5.7), respectively, as a functionof seedling age. No significant changes in enzyme levels weredetected during hypocotyl development. GDH preparations of thedifferent organs were resolved by polyacrylamide gel electrophoresisinto 2 to 4 isozymes. The isozymic pattern was influenced byseedling age and organ tested. The slowest moving isozyme (No.1) appears to be responsible for the changes in GDH levels observedin cotyledons and roots. We isolated isozyme 1 and GDH fractionchiefly containingisozyme 2, by DEAE-cellulose chromatography. GDH was purified approximately 53-fold from the particulatefraction of cotyledons. The pH optima for NADH and NAD activitieswere 8.2 and 8.9, respectively. Michaelis constants were foundto be: -ketoglutarate, 8mM; glutamate, 4 mM; ammonium, 35.4mM; NAD, 0.26 mM; NADH, 0.065 mM. Km values of isozymes 1 and2 were similar. The binding order of substrates in die reductiveamination reaction was NADH, -ketoglutarate and NH₄⁺. (Received July 17, 1972; )     

大肠杆菌苏氨酸合成途径动力学模型的构建与分析

动力学模型分析有利于理解生物系统的调控机制,从而为高效细胞工厂的理性设计提供指导。基于以往发表的相关途径动力学模型和测量的酶动力学数据,开发了大肠杆菌苏氨酸合成途径的动力学模型。模型包含从天冬氨酸至苏氨酸的合成途径及葡萄糖开始的为合成途径提供前体以及能量的代谢途径。与以往模型不同的是新模型中考虑了能量和还原力的平衡,从而使模型模拟的系统自身成为一个不需要从外界提供能量和还原力的自洽系统。模型稳态分析的结果表明PTS、G6PDH和HDH等反应对苏氨酸合成反应的通量控制系数较大,通过过表达这些反应的酶可以有效增加苏氨酸合成反应的通量。     

A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.     

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L⁻¹ culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis–Menten kinetics, exhibiting apparent mild negative co-operativity with both l -glutamate and NADP⁺, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis–Menten kinetics but both 2-oxoglutarate and NH₄⁺ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.     

p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.     

Kinetic and mechanistic analysis of the Escherichia coli ribD-encoded bifunctional deaminase-reductase involved in riboflavin biosynthesis

Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis appears to be an attractive target for drug design, since appropriate inhibitors of the pathway would selectively target the microorganism. We have cloned and solubly expressed the bifunctional ribD gene from Escherichia coli, whose three-dimensional structure was recently determined. We have demonstrated that the rate of deamination (370 min (-1)) exceeds the rate of reduction (19 min (-1)), suggesting no channeling between the two active sites. The reductive ring opening reaction occurs via a hydride transfer from the C 4- pro-R hydrogen of NADPH to C'-1 of ribose and is the rate-limiting step in the overall reaction, exhibiting a primary kinetic isotope effect ( (D) V) of 2.2. We also show that the INH-NADP adduct, one of the active forms of the anti-TB drug isoniazid, inhibits the E. coli RibD. On the basis of the observed patterns of inhibition versus the two substrates, we propose that the RibD-catalyzed reduction step follows a kinetic scheme similar to that of its structural homologue, DHFR.