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. 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

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 effect of pH

1981 ◽  
Vol 195 (3) ◽  
pp. 745-751 ◽  
Author(s):  
R B Gregory ◽  
S Ainsworth
Keyword(s):  
Pyruvate Kinase ◽  
Exponential Model ◽  
Proton Donor ◽  
Free Enzyme ◽  
Phosphoryl Group ◽  
Model Parameters ◽  
Phosphoryl Transfer ◽  
Rabbit Muscle ◽  
Ph Range ◽  
Muscle Pyruvate Kinase

The regulatory behavior of rabbit pyruvate kinase has been studied as a function of pH. The initial velocity of the enzyme-catalysed reaction as a function of ADP concentration was analysed with the exponential model for a regulatory enzyme. The analysis of the exponential model parameters as functions of pH provided pK values of 6.6 and 8.08 for the free enzyme in its fully ADP-bound conformation. By contrast, the binding of ADP to the ADP-free conformation of the free enzyme did not involve groups that ionize within the pH range (6.2-8.5) of these experiments. The results suggest that homotropic allosteric interactions actually alter the mode of ADP binding. The pK values of 6.63 and 9.00 determined from the analysis of V as a function of pH are readily interpreted in terms of a direct phosphoryl-transfer mechanism in which the beta-phosphoryl group of ADP (pK 6.63) acts as the nucleophile and a lysine epsilon-amino group (pK 9.0) acts as the proton donor in the pyruvate kinase reaction.

Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties

1982 ◽  
Vol 60 (9) ◽  
pp. 861-866
Author(s):  
Chiu-Yin Kwan ◽  
Robert C. Davis
Keyword(s):  
Amino Acids ◽  
Optical Properties ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Metal Ion ◽  
Inhibitory Effect ◽  
Rabbit Muscle ◽  
Divalent Metal Ion ◽  
Muscle Pyruvate Kinase ◽  
Difference Absorption

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)–enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenolpyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)–enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.

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