scholarly journals 3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties

1985 ◽  
Vol 227 (2) ◽  
pp. 591-599 ◽  
Author(s):  
D M Lowe ◽  
P K Tubbs
Keyword(s):  
Coenzyme A ◽  
Catalytic Properties ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Purification Procedure ◽  
Acetyl Coa ◽  
Mixed Product ◽  
Cellulose Phosphate ◽  
Ping Pong ◽  
Enzyme Intermediate

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.

scholarly journals The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

1974 ◽  
Vol 139 (1) ◽  
pp. 109-121 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Rat Liver ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Keto Form ◽  
Ping Pong ◽  
Substrate Inhibitor ◽  
The Hill ◽  
Covalent Intermediate

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg2+ inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg2+ but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a Ki of 67μm. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

scholarly journals A product-inhibition study of the mechanism of mitochondrial octanoyl-coenzyme A synthetase

1969 ◽  
Vol 111 (3) ◽  
pp. 257-262 ◽  
Author(s):  
A. B. Graham ◽  
M. V. Park
Keyword(s):  
Coenzyme A ◽  
Liver Mitochondria ◽  
Product Inhibition ◽  
Inhibition Kinetics ◽  
Inhibition Study ◽  
Ping Pong ◽  
Kinetics Of ◽  
Allosteric Activator

By a study of the product-inhibition kinetics of the octanoyl-CoA synthetase from ox liver mitochondria, evidence was obtained consistent with the hypothesis that the enzyme reacts by a Bi Uni Uni Bi Ping Pong type of mechanism in which the order of addition and evolution of substrates and products is CoA, octanoate, octanoyl-CoA, ATP, PPi and AMP. There is also evidence that more than one molecule of CoA can add to the enzyme and that it may act as an allosteric activator.

scholarly journals Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris

1981 ◽  
Vol 199 (2) ◽  
pp. 383-392 ◽  
Author(s):  
T A Walker ◽  
Z L Jonak ◽  
L M S Worsham ◽  
M L Ernst-Fonberg
Keyword(s):  
Fatty Acid ◽  
Euglena Gracilis ◽  
Substrate Inhibition ◽  
Fatty Acid Synthetase ◽  
Synthetase Activity ◽  
Multienzyme Complex ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Ping Pong ◽  
Wide Range

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

scholarly journals Asymmetric sulfoxidation of thioether catalyzed by soybean pod peroxidase to form enantiopure sulfoxide in water-in-oil microemulsions: kinetic model

2020 ◽  
Author(s):  
yuanyuan Zhang ◽  
Huiling Li ◽  
Zhiyong Wang ◽  
Depeng Li ◽  
Xin Xin Gao
Keyword(s):  
Kinetic Model ◽  
Substrate Inhibition ◽  
Model Fitting ◽  
Product Inhibition ◽  
Enzymatic Reactions ◽  
Asymmetric Oxidation ◽  
Organic Sulfides ◽  
Ping Pong ◽  
Electron Reduction ◽  
Nonenzymatic Reactions

The asymmetric sulfoxidation catalyzed by soybean pod peroxidase (SPP) in water-in-oil microemulsions were carried out with the yield of 91.56% and e.e of 96.08% at the activity of SPP of 3200 U ml-1 and 50℃ for 5 h. The mechanism with a two-electron reduction of SPP-I is accompanied with a single-electron transfer to SPP-I and nonenzymatic reactions, indicating that three concomitant sub-mechanisms contribute to the asymmetric oxidation involving five enzymatic and two nonenzymatic reactions, which can represent the asymmetric sulfoxidation of organic sulfides to form enantiopure sulfoxides. With 5.44% of the average relative deviation, a kinetic model fitting experimental data very well was developed. The enzymatic reactions may follow ping-pong mechanism with substrate inhibition of H2O2 and product inhibition of esomeprazole, while nonenzymatic reactions, a power law. Those results indicate that SPP with a lower cost and higher thermal stability may be used as an effective substitute for Horseradish Peroxidase.

scholarly journals Purification and steady-state kinetics of adenosine 5′-pyrophosphate sulphurylase from baker's yeast

1977 ◽  
Vol 165 (1) ◽  
pp. 149-155 ◽  
Author(s):  
R G Nicholls
Keyword(s):  
Exchange Reaction ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Substrate Inhibition ◽  
Deae Cellulose ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Kinetics Of

ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5′-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5′-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.

scholarly journals Asymmetric sulfoxidation of thioether catalyzed by soybean pod peroxidase ：kinetic model

2020 ◽  
Author(s):  
yuanyuan zhang ◽  
huiling Li ◽  
zhiyong Wang ◽  
depeng Li ◽  
xin Gao
Keyword(s):  
Kinetic Model ◽  
Substrate Inhibition ◽  
Model Fitting ◽  
Product Inhibition ◽  
Enzymatic Reactions ◽  
Asymmetric Oxidation ◽  
Organic Sulfides ◽  
Ping Pong ◽  
Electron Reduction ◽  
Nonenzymatic Reactions

The asymmetric sulfoxidation catalyzed by soybean pod peroxidase (SPP) in water-in-oil microemulsions were carried out with the yield of 91.56% and e.e of 96.08% at the activity of SPP of 3200 U ml-1 and 50℃ for 5 h. The mechanism with a two-electron reduction of SPP-I is accompanied with a single-electron transfer to SPP-I and nonenzymatic reactions, indicating that three concomitant sub-mechanisms contribute to the asymmetric oxidation involving five enzymatic and two nonenzymatic reactions, which can represent the asymmetric sulfoxidation of organic sulfides to form enantiopure sulfoxides. With 5.44% of the average relative deviation, a kinetic model fitting experimental data was developed. The enzymatic reactions may follow ping-pong mechanism with substrate inhibition of H2O2 and product inhibition of esomeprazole, while nonenzymatic reactions, a power law. Those results indicate that SPP with a lower cost and higher thermal stability may be used as an effective substitute for Horseradish Peroxidase.

scholarly journals The kinetic properties of human placental choline acetyltransferase

1971 ◽  
Vol 125 (3) ◽  
pp. 857-863 ◽  
Author(s):  
D. Morris ◽  
A. Maneckjee ◽  
Catherine Hebb
Keyword(s):  
Sodium Chloride ◽  
Choline Acetyltransferase ◽  
Product Inhibition ◽  
Rabbit Brain ◽  
Kinetic Properties ◽  
Forward Reaction ◽  
Acetyl Coa ◽  
No Inhibition ◽  
Ping Pong ◽  
Michaelis Constants

1. Michaelis constants for human placental choline acetyltransferase were shown to be dependent on the concentration of the second substrate present. The primary plots indicate a sequential rather than a Ping Pong mechanism and are of the same type with 300mm- and 500mm-sodium chloride. 2. Similar results have been obtained with rabbit brain choline acetyltransferase. 3. Product inhibition of the forward reaction has been studied. CoA inhibits competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibits competitively with respect to choline and non-competitively with respect to acetyl-CoA. No inhibition is given by acetylcholine when the enzyme is saturated with choline. 4. It is concluded that human placental choline acetyltransferase has an ordered mechanism of the Theorell–Chance type.

scholarly journals The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction

1981 ◽  
Vol 199 (1) ◽  
pp. 171-178 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Substrate Inhibition ◽  
Maximum Velocity ◽  
Product Inhibition ◽  
Mixed Product ◽  
Enzyme Form ◽  
Wide Range ◽  
No2 Reduction ◽  
K 12

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.

scholarly journals Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

1983 ◽  
Vol 213 (1) ◽  
pp. 179-185 ◽  
Author(s):  
J A Sharp ◽  
M R Edwards
Keyword(s):  
Binding Sites ◽  
Initial Velocity ◽  
Coenzyme A ◽  
Product Inhibition ◽  
Assay System ◽  
Inhibition Kinetics ◽  
Direct Assay ◽  
Coa Transferase ◽  
Ping Pong ◽  
Kinetics Of

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 × 10(-5) M, KSucc-CoA of 1.6 × 10(-4) M, KSucc of 5.6 × 10(-3) M and KAcAc of 6.7 × 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.

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