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.

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 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 Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii: a Coenzyme A Loop Saves Energy for Caffeate Activation

2013 ◽  
Vol 79 (6) ◽  
pp. 1942-1947 ◽  
Author(s):  
Verena Hess ◽  
José M. González ◽  
Anutthaman Parthasarathy ◽  
Wolfgang Buckel ◽  
Volker Müller
Keyword(s):  
Molecular Hydrogen ◽  
Coenzyme A ◽  
Polypeptide Chain ◽  
Acetobacterium Woodii ◽  
Content Type ◽  
Catalytic Domains ◽  
Coa Transferase ◽  
Ping Pong ◽  
Acetogenic Bacterium ◽  
Temperature Optima

ABSTRACTThe anaerobic acetogenic bacteriumAcetobacterium woodiicouples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product ofcarA, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function ofcarA, we overproduced its protein inEscherichia coliand then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparentKmvalues of 75 ± 5 μM for caffeate and 8 ± 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could usep-coumarate and ferulate but not cinnamate, sinapate, orp-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.

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.

Kinetic Studies of Phosphoribosyl-formylglycineamidine Synthetase

1972 ◽  
Vol 50 (5) ◽  
pp. 490-500 ◽  
Author(s):  
Samuel Y. Chu ◽  
J. Frank Henderson
Keyword(s):  
Ammonium Chloride ◽  
Initial Velocity ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Ping Pong ◽  
Inhibition Constants ◽  
Inhibition Studies

Initial velocity and product inhibition studies of phosphoribosyl-formylglycineamidine synthetase indicate that the reaction involves a fully ping pong mechanism in which glutamine binds to the free enzyme and glutamate is released before the addition of ATP. ADP is released, and phosphoribosyl-formylglycineamide then binds; the liberation of Pi is rapid, and phosphoribosyl-formylglycineamidine is the last product released from the enzyme. The Km values for glutamine, ATP, and phosphoribosyl-formylglycineamide are 1.1 × 10−4 M, 1.5 × 10−3 M, and 1.1 × 10−4 M, respectively. The Km value for ammonium chloride is 7.5 × 10−3 M, and the ratio of Vmax values with ammonium chloride and glutamine is 1/40. The inhibition constants for FGAM and Pi were calculated to be 1.3 × 10−4 M and 6.45 × 10−3 M, respectively.

scholarly journals Kinetics of Enantiomerically Enriched Synthesis of Solketal Esters Using Native and SBA-15 supported P. Fluorescens Lipase

2017 ◽  
Vol 38 (2) ◽  
pp. 209-215
Author(s):  
Aurelia Zniszczoł ◽  
Katarzyna Szymańska ◽  
Jacek Kocurek ◽  
Jolanta Bryjak ◽  
Krzysztof Walczak ◽  
...  
Keyword(s):  
Enantiomeric Excess ◽  
Product Inhibition ◽  
Acyl Donor ◽  
Vinyl Esters ◽  
Alkaline Lipase ◽  
Ping Pong ◽  
Native Lipase ◽  
Optimized Conditions ◽  
Kinetics Of ◽  
Positive Effect

Abstract The studies showed that alkaline lipase from Pseudomonas fluorescens enables an irreversible transesterification of vinyl esters to give enantiomeric excess (eeR) of about 80% using vinyl butyrate as acyl donor and diisopropyl ether as a solvent, at partially optimized conditions. For the native lipase the process was adequately described by a five-parameter Ping-Pong Bi Bi model for both enantiomers plus expression accounting for the formation of enzyme-acyl donor complex, but for the same lipase supported on mesoporous materials of SBA-15-Oc type, R-product inhibition also had to be taken into account. The use of hydrophobic support increased by more than two-fold the rate of the S-solketal conversion but even more that of R-solketal. Thus the immobilization of lipase had very positive effect on the process kinetics but decreased its enantioselectivity.

Kinetics of DIDS inhibition of HL-60 cell anion exchange rules out ping-pong model with slippage

1991 ◽  
Vol 260 (3) ◽  
pp. C535-C544 ◽  
Author(s):  
D. Restrepo ◽  
B. L. Cronise ◽  
R. B. Snyder ◽  
L. J. Spinelli ◽  
P. A. Knauf
Keyword(s):  
Anion Exchange ◽  
Competitive Inhibition ◽  
Disulfonic Acid ◽  
Inhibition Kinetics ◽  
Ping Pong ◽  
Kinetics Of ◽  
Simultaneous Model ◽  
Substrate Kinetics ◽  
Transport Site ◽  
Rule Out

According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.

Effects of modifiers on the kinetics of two-substrate enzyme reactions

1968 ◽  
Vol 46 (11) ◽  
pp. 1381-1396 ◽  
Author(s):  
J. Frank Henderson
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Rate Equations ◽  
Enzyme Reactions ◽  
Ping Pong ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

Steady state rate equations have been derived for ordered bi bi and ping pong bi bi reactions in which there are (a) one or two nonsubstrate modifiers, (b) two different binding sites for a single nonsubstrate modifier, (c) one or two substrates acting as modifiers, and (d) both nonsubstrate modifiers and substrates acting as modifiers. The deviation of these equations from the Michaelis–Menten equation is shown and methods are suggested by which many of these mechanisms can be distinguished experimentally.

Study on Reaction Kinetics of Lipase-Catalyzed Synthesis of Vanillyl Nonanoate in Acetone Media

2011 ◽  
Vol 233-235 ◽  
pp. 2660-2664
Author(s):  
Hui Lin Liu ◽  
Shao Liu ◽  
Xin Rong Dong ◽  
Da Ping Xie
Keyword(s):  
Kinetic Equation ◽  
Reaction Kinetics ◽  
Initial Velocity ◽  
High Performance ◽  
Velocity Versus ◽  
Vanillyl Alcohol ◽  
Novozyme 435 ◽  
Ping Pong ◽  
Kinetics Of ◽  
Methyl Nonanoate

The reaction kinetics for synthesis of vanillyl nonanoate (VN) by lipase-catalyzed transesterification of vanillyl alcohol and methyl nonanoate in acetone was investigated in this study. The reaction catalyzed by lipase was carried out as follows: A reaction mixture containing given concentration of substrates (1ml) and lipase Novozyme 435 (20mg) in acetone (1ml) was shaken at 30°C for 10min. The initial velocity of the reaction was calculated according to the concentration of VN detected by high performance liquid chromatography (HPLC), and the kinetic equation was obtained by analysis of the double reciprocal plot of initial velocity versus substrate concentration. The data calculated by the kinetic equation were basically in agreement with the experimental data with a correlation coefficient at 0.997. The inhibitory action of methanol (by-product) in the reaction was also investigated and the results indicated that the concentration of methanol influences both slope and intercept of y-axis of the double reciprocal curve, and the lines intersect on the X-axis. These results revealed that this transesterification follows the Ping-Pong reaction mechanism.

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