A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of l-alanine and fructose 1,6-bisphosphate

1986 ◽  
Vol 874 (2) ◽  
pp. 193-204 ◽  
Author(s):  
Pilar del Valle ◽  
Dolores de Arriaga ◽  
Felix Busto ◽  
Joaquín Soler
Keyword(s):  
Pyruvate Kinase ◽  
Fructose 1,6 Bisphosphate ◽  
Kinetics Of

scholarly journals The regulatory properties of yeast pyruvate kinase. Effect of fructose 1,6-bisphosphate

1986 ◽  
Vol 234 (3) ◽  
pp. 691-698 ◽  
Author(s):  
C N Morris ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Kinase ◽  
Substrate Concentration ◽  
Exponential Model ◽  
Fructose Bisphosphate ◽  
Inflected Form ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties ◽  
Kinetics Of ◽  
Binding Behaviour

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.

scholarly journals Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions

1983 ◽  
Vol 211 (3) ◽  
pp. 631-640 ◽  
Author(s):  
A Boiteux ◽  
M Markus ◽  
T Plesser ◽  
B Hess ◽  
M Malcovati
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Allosteric Site ◽  
Binding Properties ◽  
Rate Analysis ◽  
Saturation Kinetics ◽  
Ph Stat ◽  
Stat Method ◽  
Fructose 1,6 Bisphosphate ◽  
Kinetics Of

The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis. The data were analysed on the basis of a concerted model with three conformational states [Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem. J. 189, 421-433] by using a novel procedure for a computer-directed treatment of progress curves [Markus & Plesser (1976) Biochem. Soc. Trans. 4, 361-364]. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex. We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.

scholarly journals Characterization and Kinetics of Isoenzymes of Pyruvate Kinase from Developing Castor Bean Endosperm

1980 ◽  
Vol 65 (6) ◽  
pp. 1188-1193 ◽  
Author(s):  
Robert J. Ireland ◽  
Vincenzo De Luca ◽  
David T. Dennis
Keyword(s):  
Pyruvate Kinase ◽  
Castor Bean ◽  
Kinetics Of

scholarly journals Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

2018 ◽  
Author(s):  
Jamie A. Macpherson ◽  
Alina Theisen ◽  
Laura Masino ◽  
Louise Fets ◽  
Paul C. Driscoll ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
High Activity ◽  
Pyruvate Kinase ◽  
Cross Talk ◽  
Glycolytic Enzyme ◽  
Computational Framework ◽  
Allosteric Inhibitor ◽  
Fructose 1,6 Bisphosphate ◽  
Low Activity

ABSTRACTAllosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

scholarly journals Kinetics of a self-amplifying substrate cycle: ADP–ATP cycling assay

2000 ◽  
Vol 350 (1) ◽  
pp. 237-243 ◽  
Author(s):  
Edelmira VALERO ◽  
Ramón VARÓN ◽  
Francisco GARCÍA-CARMONA
Keyword(s):  
Lactate Dehydrogenase ◽  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Adenylate Kinase ◽  
Reaction Medium ◽  
Spectrophotometric Assay ◽  
Background Signal ◽  
Cyclic System ◽  
Exponential Increase ◽  
Kinetics Of

A kinetic study of an ATP–ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and l-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16pmol of ATP and/or ADP in a continuous spectrophotometric assay.

scholarly journals A kinetic study of rabbit muscle pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 223-236 ◽  
Author(s):  
S. Ainsworth ◽  
N. Macfarlane
Keyword(s):  
Reaction Mechanism ◽  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Random Order ◽  
Experimental Results ◽  
Rabbit Muscle ◽  
Dead End ◽  
Inhibition Constants ◽  
Muscle Pyruvate Kinase ◽  
Kinetics Of

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg2+, form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

Effects of phenylalanine and alanine on the kinetics of bovine pyruvate kinase isoenzymes

Biochemistry ◽  
1975 ◽  
Vol 14 (18) ◽  
pp. 4041-4045 ◽  
Author(s):  
Janet M. Cardenas ◽  
J. Jeffrey Strandholm ◽  
Joan M. Miller
Keyword(s):  
Pyruvate Kinase ◽  
Pyruvate Kinase Isoenzymes ◽  
Kinetics Of
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