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 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 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.

Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase

2001 ◽  
Vol 357 (1) ◽  
pp. 283-288 ◽  
Author(s):  
Donald A. VESSEY ◽  
Michael KELLEY
Keyword(s):  
Reaction Mechanism ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Product Inhibition ◽  
Bivalent Cation ◽  
Medium Chain ◽  
Apparent Homogeneity ◽  
Ping Pong ◽  
Inhibition Studies

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg2+ results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg2+ and ATP are varied together.

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.

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.

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