title: Yeast phosphofructokinase: pre-steady-state and stationary kinetic studies on a cross-linked enzyme form.
abstract: A cross-linked form of yeast phosphofructokinase in which up to four of the subunits of the octameric molecule were found covalently linked by dimethyl suberimidate exhibits no cooperativity with respect to fructose 6-phosphate and is only weakly inhibited by ATP. The modified enzyme is activated by AMP and fructose 2,6-bisphosphate. Both effectors abolish ATP inhibition even at low concentration of fructose 6-phosphate and increase the affinity of modified phosphofructokinase to fructose 6-phosphate but are without significant effect on the respective maximum activity. The corresponding kinetic patterns exhibit similarity to those of the native enzyme at high concentration of fructose 6-phosphate. In comparison to native phosphofructo-kinase the extent of activation by AMP and fructose 2,6-bisphosphate is of minor amount. As shown for native phosphofructokinase, the enzyme which had been cross-linked in the absence of any effector shows a lag phase of product formation. This initial transient phase completely disappears if the enzyme is modified in the presence of fructose 6-phosphate, fructose 1,6-bisphosphate, and fructose 2,6-bisphosphate, respectively. The same result is obtained by preincubation of the enzyme cross-linked in the absence of any effector with each of these fructose phosphates. The kinetic properties of the modified enzyme indicate that cooperativity with respect to fructose 6-phosphate is not a prerequisite for the allosteric modulation of enzyme activity by AMP and fructose 2,6-bisphosphate and support the idea of multiple conformational determinants in yeast phosphofructokinase. The results suggest that moderate intramolecular cross-linking can provide a simple experimental tool to stabilize different conformational states of an enzyme.
authors: Institut für Biochemie, Karl-Marx-Universität Leipzig, DDR.
authors: T
authors: T
authors: Kriegel
authors: J
authors: J
authors: Bär
authors: W
authors: W
authors: Schellenberger
authors: S
authors: S
authors: König
authors: J
authors: J
authors: Behlke
authors: G
authors: G
authors: Hübner
authors: G
authors: G
authors: Kopperschläger
mesh_terms: D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005635:Fructosediphosphates / Q000494:pharmacology; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015003:Yeasts / Q000187:drug effects / Q000201:enzymology*
chemical_list: D003432:Cross-Linking Reagents; D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1