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 Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli

1980 ◽  
Vol 189 (3) ◽  
pp. 421-433 ◽  
Author(s):  
M Markus ◽  
T Plesser ◽  
A Boiteux ◽  
B Hess ◽  
M Malcovati
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Regulatory Region ◽  
Conformational State ◽  
Type I ◽  
Allosteric Site ◽  
Rate Law ◽  
Ph Stat ◽  
Stat Method ◽  
Concentration Space

Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C). Additional initial-rate measurement were performed to assess specific points. Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves. Two models, both extensions of the concerted model given by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88–118] with four protomers, could be fitted to the data within the experimental error. Model discrimination in favour of one of these models was possible by proper experimental design. In the selected model one conformational state of the enzyme forms the active complex. The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations. In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site.

Potentiometrische Analyse der Kinetik des gegenläufigen Nettotransports von K- und H-Ionen durch die Membran von E. coli B / Potentiometric Analysis of the Kinetics of the Counter-current Net Transport of K- and H-Ions across the Membrane of E. coli B

1972 ◽  
Vol 27 (8) ◽  
pp. 996-1000 ◽  
Author(s):  
Raul L′Orange ◽  
Beate Frommer-Evers
Keyword(s):  
Bacterial Membrane ◽  
External Concentration ◽  
E Coli ◽  
Saturation Kinetics ◽  
Potentiometric Analysis ◽  
Ph Stat ◽  
Counter Current ◽  
Kinetics Of

Potassium measurements were carried out continuously with a potassiumselective electrode of the ORION type by compensation of the uptake of starved bacteria. The output of H+-ions was simultaneously registered by a pH-stat. Addition of either glucose, pyruvate or lactate triggers different iontransport velocities across the bacterial membrane. K+ obeys saturation kinetics not only referring to the external concentration of K+, but also with respect to the concentration of lactate and the same applies to the velocity of the extruded He-ions referring only to lactate.It could be demonstrated that an exchange for H+-ions is not an indispensable condition for the uptake of K+-ions by E. coli.

scholarly journals Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation–π bond

2019 ◽  
Vol 75 (6) ◽  
pp. 461-469 ◽  
Author(s):  
Jeffrey S. McFarlane ◽  
Trey A. Ronnebaum ◽  
Kathleen M. Meneely ◽  
Annemarie Chilton ◽  
Aron W. Fenton ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Functional Data ◽  
Human Liver ◽  
Phosphate Binding ◽  
Allosteric Site ◽  
Allosteric Activation ◽  
X Ray ◽  
Fructose 1,6 Bisphosphate ◽  
Open Position ◽  
Binding Loop

Human liver pyruvate kinase (hLPYK) converts phosphoenolpyruvate to pyruvate in the final step of glycolysis. hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP). The allosteric site, as defined by previous structural studies, is located in domain C between the phosphate-binding loop (residues 444–449) and the allosteric loop (residues 527–533). In this study, the X-ray crystal structures of four hLPYK variants were solved to make structural correlations with existing functional data. The variants are D499N, W527H, Δ529/S531G (called GGG here) and S531E. The results revealed a conformational toggle between the open and closed positions of the allosteric loop. In the absence of Fru-1,6-BP the open position is stabilized, in part, by a cation–π bond between Trp527 and Arg538′ (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6′-phosphate of Fru-1,6-BP in the allosteric site, leading to partial allosteric activation. Finally, the structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant.

scholarly journals An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas

1977 ◽  
Vol 165 (1) ◽  
pp. 97-105 ◽  
Author(s):  
I G Giles ◽  
P C Poat ◽  
K A Munday
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Enzyme Preparation ◽  
Carcinus Maenas ◽  
Maximal Activity ◽  
Initial Increase ◽  
Low Concentration ◽  
Saturation Kinetics ◽  
Fructose 1,6 Bisphosphate ◽  
Allosteric Activator

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.

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 Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin

2003 ◽  
Vol 185 (14) ◽  
pp. 4233-4242 ◽  
Author(s):  
Athena Sudom ◽  
Robert Walters ◽  
Landon Pastushok ◽  
Douglas Goldie ◽  
Lata Prasad ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Catalytic Mechanism ◽  
Stop Codon ◽  
Allosteric Site ◽  
C Terminus ◽  
Allosteric Regulator ◽  
Kinetics Of ◽  
Resolution Structure

ABSTRACT The 1.8-Å resolution structure of the ATP-Mg2+-Ca2+-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg2+-Mn2+-pyruvate-PCK, except for the Ca2+ and Mn2+ binding sites. Ca2+ was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca2+ bound only at the active site, indicating that there is likely no surface allosteric site. 45Ca2+ bound to PCK with a K d of 85 μM and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca2+. Separate roles of Mg2+, which binds the nucleotide, and Ca2+, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca2+-bound structure. Partial trypsin digestion abolishes Ca2+ activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca2+ binding site, probably stabilizing the C terminus. Phe409Ala, ΔPhe409, Phe413Ala, Δ397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca2+ site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

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