scholarly journals A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans

1976 ◽  
Vol 157 (1) ◽  
pp. 197-205 ◽  
Author(s):  
D F Brook ◽  
P J Large
Keyword(s):  
Steady State ◽  
Affinity Chromatography ◽  
Secondary Amine ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
State Kinetic

1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.

scholarly journals Steady-state kinetic analysis of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath)

1986 ◽  
Vol 236 (1) ◽  
pp. 155-162 ◽  
Author(s):  
J Green ◽  
H Dalton
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Product Inhibition ◽  
Methylococcus Capsulatus ◽  
Reaction Sequence ◽  
Oxidation Of Methane ◽  
Steady State Kinetic ◽  
Substitution Mechanism ◽  
Inhibition Studies ◽  
State Kinetic

A steady-state kinetic analysis of purified soluble methane mono-oxygenase of Methylococcus capsulatus (Bath) was performed. The enzyme was found to follow a concerted-substitution mechanism. Methane binds to the enzyme followed by NADH, which reacts to yield reduced enzyme and NAD+. The reduced enzyme-methane complex binds O2 to give a second ternary complex, which breaks down to release water and methanol. In this way the enzyme can control the supply of electrons to the active site to coincide with the arrival of methane. Product-inhibition studies (with propylene as substrate) supported the reaction mechanism proposed. Ki values for NAD+ and propylene oxide are reported. The Km for NADH varied from 25 microM to 300 microM, depending on the nature of the hydrocarbon substrate, and thus supports the proposed reaction sequence. With methane as substrate the Km values for methane, NADH and O2 were shown to be 3 microM, 55.8 microM and 16.8 microM respectively. With propylene as substrate the Km values for propylene, NADH and O2 were 0.94 microM, 25.2 microM and 12.7-15.9 microM respectively. Methane mono-oxygenase was shown to be well adapted to the oxidation of methane compared with other straight-chain alkanes.

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 Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

Pre-steady-state kinetic studies of rat kidneyγ-glutamyl transpeptidase confirm its ping-pong mechanism

2004 ◽  
Vol 17 (67) ◽  
pp. 529-536 ◽  
Author(s):  
Jeffrey W. Keillor ◽  
Annie Ménard ◽  
Roselyne Castonguay ◽  
Christian Lherbet ◽  
Caroline Rivard
Keyword(s):  
Steady State ◽  
Kinetic Studies ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Glutamyl Transpeptidase ◽  
State Kinetic

scholarly journals Steady-State Kinetic Analysis of Phosphotransacetylase from Methanosarcina thermophila

2006 ◽  
Vol 188 (3) ◽  
pp. 1155-1158 ◽  
Author(s):  
Sarah H. Lawrence ◽  
James G. Ferry
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Methanosarcina Thermophila ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Acetyl Group ◽  
State Kinetic ◽  
Reversible Transfer

ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.

scholarly journals Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae

1978 ◽  
Vol 173 (3) ◽  
pp. 787-798 ◽  
Author(s):  
K A Bostian ◽  
G F Betts
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Product Inhibition ◽  
Maximum Activity ◽  
Alternative Substrate ◽  
Steady State Kinetic ◽  
Direct Binding ◽  
Inhibition Studies ◽  
State Kinetic ◽  
Similar Kinetic ◽  
Substrate Addition

Data from steady-state kinetic analysis of yeast K+-activated aldehyde dehydrogenase are consistent with a ternary complex mechanism. Evidence from alternative substrate analysis and product-inhibition studies supports an ordered sequence of substrate binding in which NAD+ is the leading substrate. A preincubation requirement for NAD+ for maximum activity is also consistent with the importance of a binary enzyme-NAD+ complex. Dissociation constant for enzyme-NAD+ complex determined kinetically is in reasonable agreement with that determined by direct binding. The order of substrate addition proposed here differs from that proposed for a yeast aldehyde dehydrogenase previously reported. Different methods of purification produced an enzyme that showed similar kinetic characteristics to those reported here.

Bovine Thyroid Microsomal Monoamine Oxidase

1972 ◽  
Vol 50 (10) ◽  
pp. 1035-1047 ◽  
Author(s):  
Isa K. Mushahwar ◽  
Leo Oliner ◽  
Arthur R. Schulz
Keyword(s):  
Monoamine Oxidase ◽  
Product Inhibition ◽  
Mitochondrial Enzyme ◽  
Microsomal Enzymes ◽  
Steady State Kinetic ◽  
Noncompetitive Inhibitor ◽  
Bovine Thyroid ◽  
High Degree ◽  
Inhibition Studies ◽  
State Kinetic

Monoamine oxidase has been isolated and purified from bovine thyroid microsomes. The general characteristics and steady-state kinetic behavior of the microsomal enzyme have been compared with those of the enzyme isolated from bovine thyroid mitochondria. The enzymes from the two sources exhibit a high degree of substrate specificity with respect to the amines oxidized. 3-Iodotyramine is a noncompetitive inhibitor of tyramine oxidation in the case of both the mitochondrial and microsomal enzymes. Product inhibition studies suggest that the enzymes from mitochondria and microsomes catalyze reactions which proceed by a similar pathway. In contrast to the mitochondrial enzyme, the enzyme isolated from microsomes is susceptible to inhibition by anions in the following order; [Formula: see text].

scholarly journals The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

1973 ◽  
Vol 135 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Brian Gillham
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Glutathione S Transferase ◽  
Michaelis Constant ◽  
Dead End ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).

scholarly journals Steady-state kinetic analysis of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans

1989 ◽  
Vol 261 (1) ◽  
pp. 107-111 ◽  
Author(s):  
V L Davidson
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Paracoccus Denitrificans ◽  
Methylamine Dehydrogenase ◽  
Prosthetic Group ◽  
Ph Optimum ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Km Value ◽  
State Kinetic

A steady-state kinetic analysis was performed of the reaction of methylamine and phenazine ethosulphate (PES) with the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. Experiments with methylamine and PES as varied-concentration substrates produced a series of parallel reciprocal plots, and when the concentrations of these substrates were varied in a constant ratio a linear reciprocal plot of initial velocity against PES concentration was obtained. Nearly identical values of V/Km of PES were obtained with four different n-alkylamines. These data suggest that this reaction proceeds by a ping-pong type of mechanism. The enzyme reacted with a variety of n-alkylamines but not with secondary, tertiary or aromatic amines or amino acids. The substrate specificity was dictated primarily by the Km value exhibited by the particular amine. A deuterium kinetic isotope effect was observed with deuterated methylamine as a substrate. The enzyme exhibited a pH optimum for V at pH 7.5. The absorbance spectrum of the pyrroloquinoline quinone prosthetic group of this enzyme was also effected by pH at values greater than 7.5. The enzyme was relatively insensitive to changes in ionic strength, and exhibited a linear Arrhenius plot over a range of temperatures from 10 degrees C to 50 degrees C with an energy of activation 46 kJ/mol (11 kcal/mol).

scholarly journals Some kinetic properties of the tryptophan-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

1981 ◽  
Vol 199 (3) ◽  
pp. 657-665 ◽  
Author(s):  
G A Nimmo ◽  
J R Coggins
Keyword(s):  
Steady State ◽  
Neurospora Crassa ◽  
Kinetic Properties ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Rapid Equilibrium ◽  
State Kinetic ◽  
Phosphate Synthase

The steady-state kinetic properties of purified tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa were examined. The results suggest that the enzyme obeys a Rapid-Equilibrium Ordered mechanism, in which phosphoenolpyruvate is the first substrate to bind and 3-deoxy-D-arabino-heptulosonate 7-phosphate is the second product to be released, rather than a Ping Pong mechanism as has been reported previously. The inhibition by tryptophan was found to be parabolic competitive with respect to D-erythrose 4-phosphate and parabolic non-competitive with respect to phosphoenolpyruvate. The enzyme was inactivated by EDTA, and could be protected against this inactivation by phosphoenolpyruvate or 3-deoxy-D-arabino-heptulosonate 7-phosphate but not by D-erythrose 4-phosphate, tryptophan or Pi. This suggests that the enzyme may be a metalloenzyme.

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