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 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.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

scholarly journals The kinetic mechanism of the major form of ox kidney aldehyde reductase with d-glucuronic acid

1982 ◽  
Vol 205 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ann K. Daly ◽  
Timothy J. Mantle
Keyword(s):  
Glucuronic Acid ◽  
Alternative Pathway ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Aldehyde Reductase ◽  
Major Form ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Inhibition Studies ◽  
Kinetics Of

The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

Kinetics of the Isotope Exchange Reaction of Fluorine with Hydrogen Fluoride

1955 ◽  
Vol 23 (9) ◽  
pp. 1622-1624 ◽  
Author(s):  
Richard M. Adams ◽  
Richard B. Bernstein ◽  
Joseph J. Katz
Keyword(s):  
Exchange Reaction ◽  
Hydrogen Fluoride ◽  
Isotope Exchange ◽  
Isotope Exchange Reaction ◽  
Kinetics Of

scholarly journals Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

Succinyl Coenzyme A Synthetase of Escherichia coli: Initial Rate Kinetics of Succinyl-CoA Cleavage and Isotope Exchange Studies

1973 ◽  
Vol 51 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Frank J. Moffet ◽  
W. A. Bridger
Keyword(s):  
Isotope Exchange ◽  
Coenzyme A ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Constant Ratio ◽  
Rate Kinetics ◽  
Kinetics Of ◽  
Succinyl Coenzyme A Synthetase

Initial rate kinetic studies of succinyl coenzyme A synthetase of E. coli in the direction of succinyl-CoA cleavage are consistent with the operation of a partially random sequential kinetic mechanism with initial binding of ADP followed by random association of succinyl-CoA and Pi. The mechanism is analogous to that proposed previously for the succinyl-CoA formation reaction, and thus the kinetic mechanism of the overall reversible succinyl-CoA synthetase reaction appears to be symmetrical.Studies of the kinetics of [Formula: see text] isotope exchange at equilibrium show that this partially random sequential kinetic mechanism is not an exclusive pathway. [Formula: see text] isotope exchange rates did not show complete substrate inhibition when CoA or succinate was varied in constant ratio with Pi. However, when CoA or succinate was varied in constant ratio with succinyl-CoA, nearly complete substrate inhibition was observed. These results can be interpreted in terms of a wide variety of minor pathways of substrate binding and product release available to the enzyme under various conditions.

scholarly journals The steady-state kinetics of isotope exchange at equilibrium: one substrate–one product enzymic mechanisms where two molecules of substrate or product are bound to an enzyme molecule (Short Communication)

1974 ◽  
Vol 143 (3) ◽  
pp. 783-784
Author(s):  
Ivan G. Darvey
Keyword(s):  
Steady State ◽  
Isotope Exchange ◽  
Short Communication ◽  
Enzyme Molecule ◽  
First Order ◽  
Steady State Kinetics ◽  
The One ◽  
Kinetics Of

The conclusion that the steady-state kinetics of isotope exchange at equilibrium do not show first-order behaviour for some one substrate–one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time was shown to be erroneous.

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