scholarly journals Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea

1989 ◽  
Vol 67 (11-12) ◽  
pp. 763-769 ◽  
Author(s):  
Emidio De Carolis ◽  
Ragai K. Ibrahim
Keyword(s):  
Competitive Inhibition ◽  
Divalent Cations ◽  
Product Inhibition ◽  
Affinity Column ◽  
Ph Optimum ◽  
Substrate Interaction ◽  
Interaction Kinetics ◽  
Molecular Masses ◽  
Inhibition Studies ◽  
Kinetics Of

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.

scholarly journals The kinetic mechanism of the major form of ox kidney aldehyde reductase with d-glucuronic acid

1982 ◽  
Vol 205 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ann K. Daly ◽  
Timothy J. Mantle
Keyword(s):  
Glucuronic Acid ◽  
Alternative Pathway ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Aldehyde Reductase ◽  
Major Form ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Inhibition Studies ◽  
Kinetics Of

The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

Phase-plane geometries in enzyme kinetics

1994 ◽  
Vol 72 (3) ◽  
pp. 800-812 ◽  
Author(s):  
Simon J. Fraser ◽  
Marc R. Roussel
Keyword(s):  
Steady State ◽  
Phase Plane ◽  
Competitive Inhibition ◽  
Three Dimensional ◽  
Product Inhibition ◽  
Slow Manifold ◽  
Dimensional System ◽  
Planar System ◽  
State Behaviour ◽  
Kinetics Of

The transient and steady-state behaviour of the reversible Michaelis–Menten mechanism [R] and Competitive Inhibition (CI) mechanism is studied by analysis in the phase plane. Usually, the kinetics of both mechanisms is simplified to give a modified Michaelis–Menten velocity expression; this applies to the CI mechanism with excess inhibitor and to mechanism [R] in the product inhibition limit. In this paper, [R] is treated exactly as a plane autonomous system of differential equations and its true (dynamical) steady state is described by a line-like slow manifold M. Initial velocity experiments for [R] no longer strictly correspond to the hyperbolic law (as in the irreversible Michaelis–Menten mechanism) and this leads to corrections to the standard integrated rate law. Using a new analysis, the slow dynamics of the CI mechanism is reduced from a three-dimensional system to a planar system. In this mechanism transient decay collapses the trajectory flow onto a two-dimensional "slow" surface Σ; motion on Σ can be treated exactly as projected dynamics in the plane. This projected flow may differ in important ways from that of two-step mechanisms, e.g., it may lack a proper steady state. The relevance of these more accurate dynamical descriptions is discussed in relation to experimental design and metabolic function.

scholarly journals Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate

1986 ◽  
Vol 234 (2) ◽  
pp. 317-323 ◽  
Author(s):  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Isocitrate Dehydrogenase ◽  
Initial Rate ◽  
Potent Inhibitor ◽  
Competitive Inhibition ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Coenzyme Binding ◽  
Inhibition Studies

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.

scholarly journals Artemia purine phosphoribosyltransferases. Purification and characterization

1991 ◽  
Vol 275 (2) ◽  
pp. 327-334 ◽  
Author(s):  
C Montero ◽  
P Llorente
Keyword(s):  
Divalent Cations ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Ph Profile ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Artemia Cysts ◽  
Inhibition Studies

Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.

scholarly journals The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

2003 ◽  
Vol 69 (6) ◽  
pp. 3272-3279 ◽  
Author(s):  
Nuno Empadinhas ◽  
Luciana Albuquerque ◽  
Anke Henne ◽  
Helena Santos ◽  
Milton S. da Costa
Keyword(s):  
Biosynthetic Pathway ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Hyperthermophilic Archaea ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Homologous Genes ◽  
Molecular Masses

ABSTRACT The biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90�C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95�C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn2+ was most effective for MPGS, while Mn2+, Co2+, Mg2+, and to a lesser extent Ni2+ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.

scholarly journals Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites

1973 ◽  
Vol 131 (3) ◽  
pp. 451-458 ◽  
Author(s):  
K. F. Wong ◽  
D. D. Davies
Keyword(s):  
Zea Mays ◽  
Phosphoenolpyruvate Carboxylase ◽  
Competitive Inhibition ◽  
Energy Charge ◽  
Dicarboxylic Acids ◽  
Ph Optimum ◽  
Km Value ◽  
Sugar Phosphates ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the Km value for phosphoenolpyruvate is 80μm. When assayed at 30°C, the enzyme shows normal Michaelis–Menten kinetics, but when assayed at 45°C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by Pi, by SO42- and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.

Purine catabolism in man: inhibition of 5′-phosphomonoesterase activities from placental microsomes

1976 ◽  
Vol 54 (12) ◽  
pp. 1055-1060 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Alkaline Phosphatase ◽  
Product Inhibition ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phosphomonoesterase Activity ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Mechanism Of Interaction ◽  
Noncompetitive Inhibitor ◽  
Inhibition Studies

The 5′-phosphomonoesterase activity of 5′-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) participates in the catabolism of purine ribonucleotides to uric acid in humans.Initial velocity studies of 5′-nucleotidase suggest a sequential mechanism of interaction between AMP and MgCl2, with a Km of 14 and 3 μM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhibitors when AMP was the substrate, with a Ki slope of 10, 20, or 54 μM, respectively. GTP was a noncompetitive inhibitor, with a Ki slope of 120 μM.The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2.These observations suggest that 5′-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5′-nucleotidase.

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