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.

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 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.

Steady-state kinetic analysis of triacylglycerol delivery into mesenteric lymph

1986 ◽  
Vol 251 (2) ◽  
pp. G263-G269 ◽  
Author(s):  
C. M. Mansbach ◽  
A. Arnold
Keyword(s):  
Steady State ◽  
Turnover Rate ◽  
Specific Activity ◽  
Direct Proportion ◽  
Precursor Pool ◽  
Mesenteric Lymph ◽  
Steady State Kinetic ◽  
Glyceryl Trioleate ◽  
State Kinetic ◽  
Fractional Turnover

The output of triacylglycerol in chylomicrons can be increased 60% by prefeeding rats with a 20% fat diet or 110% by including phosphatidylcholine in a lipid infusion. The present study was designed to determine whether the increment was due to an expansion of the chylomicron triacylglycerol precursor pool or an increase in its fractional turnover rate. A steady-state kinetic model was established in rats receiving 135 mumol glyceryl trioleate/h. The decay in specific activity of triacylglycerol after removal of radiolabeled glyceryl trioleate from the duodenal infusate was followed for 4 h and analyzed by the SAAM 23 program. It was found that the fractional turnover rate of the chylomicron precursor pool remained the same in each experimental condition. However, the pool was found to expand in direct proportion to the chylomicron triacylglycerol output. Functionally the infused [3H]glyceride-glycerol and tri[14C]oleate behaved the same in lymph chylomicrons and was 90% of infusate specific activity. In summary, these data suggest that increases in chylomicron triacylglycerol output are dependent on the size of the mucosal precursor pool and the monoacylglycerol acyltransferase synthetic pathway for its triacylglycerol.

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