Substrate Inhibition of Pyruvate Kinase Isolated from the Leg Muscle of Carcinus maenas

1976 ◽  
Vol 4 (6) ◽  
pp. 1158-1160 ◽  
Author(s):  
COLIN J. NEWTON ◽  
PETER C. POAT ◽  
KENNETH A. MUNDAY
Keyword(s):  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Carcinus Maenas ◽  
Leg Muscle

scholarly journals A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas

1980 ◽  
Vol 185 (2) ◽  
pp. 289-299 ◽  
Author(s):  
I G Giles ◽  
P C Poat
Keyword(s):  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Carcinus Maenas ◽  
Product Inhibition ◽  
Dead End ◽  
Steady State Kinetic ◽  
Competitive Substrate ◽  
Fructose 1,6 Bisphosphate ◽  
Inhibition Studies ◽  
State Kinetic

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

Comparative study of free and bound glycolytic enzymes from sea scorpion brain

1998 ◽  
Vol 76 (4) ◽  
pp. 609-614 ◽  
Author(s):  
Volodymyr I Lushchak ◽  
Ljudmyla P Lushchak ◽  
Tetjana V Bahnjukova ◽  
Alexei V Spichenkov ◽  
Kenneth B Storey
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Physiological Role ◽  
Glycolytic Enzymes ◽  
Free Form ◽  
Hill Coefficients ◽  
Low Ionic Strength ◽  
Scorpaena Porcus

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45°C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.Key words: hexokinase, lactate dehydrogenase, pyruvate kinase, enzyme binding, Scorpaena porcus.

Regulation of Pyruvate Kinase from the Hepatopancreas of the Crab Carcinus maenas

1975 ◽  
Vol 3 (3) ◽  
pp. 400-402 ◽  
Author(s):  
IAN G. GILES ◽  
PETER C. POAT ◽  
KENNETH A. MUNDAY
Keyword(s):  
Pyruvate Kinase ◽  
Carcinus Maenas

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.

A Kinetic Study of Muscle Pyruvate Kinase from Carcinus maenas

1976 ◽  
Vol 4 (3) ◽  
pp. 479-481 ◽  
Author(s):  
COLIN J. NEWTON ◽  
PETER C. POAT ◽  
KENNETH A. MUNDAY
Keyword(s):  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Carcinus Maenas ◽  
Muscle Pyruvate Kinase

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.

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