Kinetic studies on the regulation of rabbit liver pyruvate kinase.
Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D005636:Fructosephosphates / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006598:Hexosediphosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D005636:Fructosephosphates; D006598:Hexosediphosphates; D007527:Isoenzymes; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium; D000409:Alanine; D011188:Potassium