scholarly journals Kinetics of carbonyl reductase from human brain

1987 ◽  
Vol 244 (1) ◽  
pp. 165-171 ◽  
Author(s):  
K M Bohren ◽  
J P von Wartburg ◽  
B Wermuth
Keyword(s):  
Isotope Effects ◽  
Product Inhibition ◽  
Carbonyl Reductase ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Rate Analysis ◽  
Sequential Mechanism ◽  
Straight Lines ◽  
Inhibition Studies ◽  
Kinetics Of

Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.

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.

scholarly journals Purification and kinetic properties of ox brain histamine N-methyltransferase

1986 ◽  
Vol 233 (3) ◽  
pp. 669-676 ◽  
Author(s):  
W L Gitomer ◽  
K F Tipton
Keyword(s):  
Acetic Acid ◽  
Steady State ◽  
Mouse Brain ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Brain Histamine ◽  
Inhibition Studies

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

scholarly journals Kinetic mechanism of pulmonary carbonyl reductase

1988 ◽  
Vol 252 (1) ◽  
pp. 17-22 ◽  
Author(s):  
K Matsuura ◽  
T Nakayama ◽  
M Nakagawa ◽  
A Hara ◽  
H Sawada
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Carbonyl Reductase ◽  
Ternary Complexes ◽  
Enzyme Mechanism ◽  
The Other ◽  
Binding Studies ◽  
Forward Reaction ◽  
Cibacron Blue ◽  
Inhibition Studies

The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes.

scholarly journals Kinetic properties of spermine synthase from bovine brain

1983 ◽  
Vol 215 (3) ◽  
pp. 669-676 ◽  
Author(s):  
R L Pajula
Keyword(s):  
Initial Velocity ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Bovine Brain ◽  
Kinetic Properties ◽  
Methylthioadenosine Phosphorylase ◽  
Spermine Synthase ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Competitive Product

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5′-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5′-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5′-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

Molecular Forms and Kinetic Properties of Pyruvate, PI Dikinase From Two Populations of Barnyard Grass (Echinochloa crus-galli) From Sites of Contrasting Climates

1996 ◽  
Vol 23 (2) ◽  
pp. 191 ◽  
Author(s):  
JP Simon
Keyword(s):  
Specific Activity ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Activity Levels ◽  
Light Activation ◽  
Turnover Number ◽  
Barnyard Grass ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Two Populations

Plants from two populations of the C4 barnyard grass (Echinochloa crus-galli (L.) Beauv.) from Qu�bec (QUE) and Mississippi (MISS) were acclimated under controlled conditions to 26/20 and 14/8�C daylnight. The apparent energy of activation (Ea, Km for pyruvate, Vmax/Km ratios, Kcat (substrate turnover number) and specific activity of pyruvate, PI dikinase (PPDK, EC 2.7.9.1) were analysed from partially purified Sephadex G-25 extracts of PPDK from leaves and from highly purified PPDK. PPDK from both populations consisted of one isomorph with the same electrophoretic mobility in polyacrylamide gels and similar molecular weights for the native enzyme (385 kDa) and for the subunit of the tetramer (94.8 kDa). No significant differences were observed for any of the kinetic properties of partially purified or purified PPDK or for the specific activity per mg protein of purified PPDK extracted from plants of the two populations and acclimated to the two thermoperiods. Net photosynthetic rates (Ps) were positively correlated with PPDK activity levels (E) but ElPs ratios were lower than 1.0, ranging from 0.43 to 0.67. Results indicate that differences in activity levels, thermal properties and in the kinetics of light activation and dark inactivation of PPDK extracted from cold-acclimated MISS and QUE plants, as reported in earlier studies, are due to causes other than kinetic properties or electrophoretic characteristics of PPDK.

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.

Glutamate Dehydrogenase from Medicago sativa L., Purification and Comparative Kinetic Studies of the Organ-Specific Multiple Forms

1980 ◽  
Vol 35 (5-6) ◽  
pp. 406-415 ◽  
Author(s):  
Marianne Nagel ◽  
Hartmann
Keyword(s):  
Medicago Sativa ◽  
Glutamate Dehydrogenase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Enzyme Preparations ◽  
Organ Specific ◽  
Nad Oxidoreductase ◽  
Medicago Sativa L ◽  
Inhibition Studies

Abstract NAD-specific glutamate dehydrogenase [L-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-pattems of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measure­ ments and product inhibition studies are consistent with an ordered ternary-binary kinetic mecha­ nism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.

scholarly journals Kinetic studies with the low-Km aldehyde reductase from ox brain

1985 ◽  
Vol 227 (2) ◽  
pp. 621-627 ◽  
Author(s):  
C M Ryle ◽  
K F Tipton
Keyword(s):  
Initial Velocity ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Binary Complex ◽  
Aldehyde Reductase ◽  
Apparent Dissociation Constant ◽  
Displacement Mechanism ◽  
Sequential Mechanism ◽  
Inhibition Studies

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

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