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 The regulatory properties of yeast pyruvate kinase. Effect of pH

1986 ◽  
Vol 234 (3) ◽  
pp. 699-703 ◽  
Author(s):  
J Kinderlerer ◽  
S Ainsworth ◽  
C N Morris ◽  
N Rhodes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Kinase ◽  
Exponential Model ◽  
Effect Of Ph ◽  
Ph Range ◽  
Regulatory Properties ◽  
Kinetics Of

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the effector H+ in the pH range 5-6.6. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that the data could be described by the exponential model for a regulatory enzyme. On that basis, it was concluded that the binding of H+ is positively interactive and that the protonated enzyme is catalytically inactive. It was also found that H+ interacts positively with phosphoenolpyruvate but negatively with both ADP and Mg2+.

scholarly journals The regulatory properties of yeast pyruvate kinase

1984 ◽  
Vol 217 (3) ◽  
pp. 641-647 ◽  
Author(s):  
C N Morris ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
Exponential Model ◽  
Positive Interactions ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Catalysed Reaction ◽  
Regulatory Properties ◽  
Kinetics Of ◽  
Binding Behaviour

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.

scholarly journals The regulatory properties of rabbit muscle pyruvate kinase. The influence of effector concentrations

1983 ◽  
Vol 209 (2) ◽  
pp. 413-415 ◽  
Author(s):  
R B Gregory ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Pyruvate Kinase ◽  
Initial Velocity ◽  
Exponential Model ◽  
Rabbit Muscle ◽  
Muscle Pyruvate Kinase ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

The initial velocity of the reaction catalysed by rabbit muscle pyruvate kinase was studied as a function of the concentrations of the modifiers phenylalanine and fructose 1,6-bisphosphate under conditions where the relationships between the initial velocities and the concentrations of substrates are non-hyperbolic. It is shown that these data can be represented by the exponential model for a regulatory enzyme.

scholarly journals The regulatory properties of rabbit muscle pyruvate kinase. The influence of substrate concentrations

1983 ◽  
Vol 209 (2) ◽  
pp. 401-411 ◽  
Author(s):  
S Ainsworth ◽  
J Kinderlerer ◽  
R B Gregory
Keyword(s):  
Pyruvate Kinase ◽  
Exponential Model ◽  
Rabbit Muscle ◽  
Catalysed Reaction ◽  
Dead End ◽  
Influence Of Substrate ◽  
Muscle Pyruvate Kinase ◽  
Regulatory Properties ◽  
Kinetics Of ◽  
Substrate Concentrations

The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.

scholarly journals The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations

1986 ◽  
Vol 234 (3) ◽  
pp. 705-715 ◽  
Author(s):  
N Rhodes ◽  
C N Morris ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Pyruvate Kinase ◽  
Dissociation Constants ◽  
Exponential Model ◽  
Positive Interactions ◽  
Data Sets ◽  
Positive Interaction ◽  
Simultaneous Activation ◽  
Regulatory Properties ◽  
Kinetics Of

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.

On the Role of Magnesium in the Reaction of the Pyruvate Kinase from Salmonella typhimurium

1986 ◽  
Vol 41 (11-12) ◽  
pp. 1018-1022
Author(s):  
Concepcion Garcia-Olalla ◽  
Amando Garrido-Pertierra
Keyword(s):  
Salmonella Typhimurium ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Physiological Significance ◽  
Hill Coefficient ◽  
Magnesium Concentration ◽  
Pyruvate Kinases ◽  
Kinetics Of ◽  
Binding Behaviour

Abstract The kinetics of the two purified forms of pyruvate kinase from Salmonella typhimurium LT-2 were studied in assays at pH 6.8 where the relationships between the initial velocities of the catalysed reactions and Mg2+ are non-hyperbolic. The analysis show that Mg2+ display positive homotropic interactions in their binding behaviour with Hill coefficient values of 2.5 and 1.2 for the form I and II, respectively. The binding sites of the cation to the pyruvate kinases seem to be independent to those for phosphoenolpyruvate and adenosine 5′-diphosphate; changes in the magnesium concentration might be of physiological significance in relation to a rapid regeneration of adenosine 5′-triphosphate by means of the pyruvate kinase reaction.

Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties

1988 ◽  
Vol 66 (2) ◽  
pp. 148-157 ◽  
Author(s):  
Félix Busto ◽  
Pilar Del Valle ◽  
Joaquín Soler
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Kinetic Properties ◽  
Saturation Curve ◽  
Phycomyces Blakesleeanus ◽  
Competitive Inhibitors ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) was purified approximately 500-fold to a final specific activity of 25 U∙mg protein−1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces requires Mg2+ for activity, but not K+ or NH4. It showed a transition temperature at 36 °C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curves to a hyperboolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type 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.

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