title: Kinetic studies on the regulation of rabbit liver pyruvate kinase.
abstract: 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+).
mesh_terms: 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
chemical_list: 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