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 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.

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 A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas

1980 ◽  
Vol 185 (2) ◽  
pp. 289-299 ◽  
Author(s):  
I G Giles ◽  
P C Poat
Keyword(s):  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Carcinus Maenas ◽  
Product Inhibition ◽  
Dead End ◽  
Steady State Kinetic ◽  
Competitive Substrate ◽  
Fructose 1,6 Bisphosphate ◽  
Inhibition Studies ◽  
State Kinetic

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

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.

Steady-State Kinetic and Inhibition Studies of the Mammalian Target of Rapamycin (mTOR) Kinase Domain and mTOR Complexes

Biochemistry ◽  
2010 ◽  
Vol 49 (39) ◽  
pp. 8488-8498 ◽  
Author(s):  
Zhihua Tao ◽  
John Barker ◽  
Stone D.-H. Shi ◽  
Michael Gehring ◽  
Shaoxian Sun
Keyword(s):  
Steady State ◽  
Mammalian Target Of Rapamycin ◽  
Kinase Domain ◽  
Target Of Rapamycin ◽  
Mtor Kinase ◽  
Steady State Kinetic ◽  
Inhibition Studies ◽  
State Kinetic

scholarly journals Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase

1992 ◽  
Vol 286 (1) ◽  
pp. 243-251 ◽  
Author(s):  
N L Deans ◽  
R D Allison ◽  
D L Purich
Keyword(s):  
Steady State ◽  
Thermal Inactivation ◽  
Enzyme Reaction ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Maximal Velocity ◽  
Steady State Kinetic ◽  
Tubulin Tyrosine Ligase ◽  
State Kinetic

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.

scholarly journals The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme

1985 ◽  
Vol 225 (2) ◽  
pp. 335-342 ◽  
Author(s):  
V J Davisson ◽  
A R Schulz
Keyword(s):  
Steady State ◽  
Malic Enzyme ◽  
Specific Activity ◽  
Rabbit Heart ◽  
Product Inhibition ◽  
Saturation Curve ◽  
Steady State Kinetic ◽  
Sequential Binding ◽  
State Kinetic ◽  
Reductive Carboxylation

The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate: NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrate describes this inhibition. The apparent kinetic parameters for this reaction are Ka(NADH) = 239 microM and Vr = 1.1 mumol/min per mg at 23 degrees C. The steady-state product-inhibition patterns for pyruvate and NADH indicate a sequential binding of the substrates: NAD+ followed by L-malate. These data also indicate that NADH is the last product released. A steady-state kinetic model is proposed that incorporates NADH-enzyme dead-end complexes.

Sign in / Sign up

Export Citation Format

Share Document