Yeast phosphofructokinase: kinetic characterization of a substrate-imprinted enzyme conformation.
Intramolecular cross-linking of octameric yeast phosphofructokinase was applied to study the effect of substrate-imprinted conformational changes on the regulatory properties of the enzyme:Cross-linking performed in the presence of fructose 6-phosphate yields a substrate-imprinted enzyme species the affinity of which towards this substrate is significantly higher than that of native phosphofructokinase and of the enzyme cross-linked in the absence of fructose 6-phosphate. The enzyme cross-linked in the presence of fructose 6-phosphate does not exhibit cooperativity with respect to this substrate but is still activated by AMP and by fructose 2,6-bisphosphate. This activation consists in an increase of substrate affinity with respect to fructose 6-phosphate. In the absence of positive effectors, the maximum activity of the cross-linked enzyme corresponds to the respective values of native phosphofructokinase when activated by AMP or by fructose 2,6-bisphosphate. At saturating levels of AMP and of fructose 2,6-bisphosphate, nearly identical affinities with respect to fructose 6-phosphate are found, ranging between the Km values of native phosphofructokinase activated by AMP and by fructose 2,6-bisphosphate. Covalent stabilization of the substrate-imprinted enzyme conformation does not affect the interaction of phosphofructokinase with ATP at the substrate-binding site. The results suggest that the allosteric regulation of yeast phosphofructokinase is mainly related to conformational changes controlled by fructose 6-phosphate while the ATP affinity at the catalytic site of the enzyme remains essentially unaffected.
Kriegel
T
T
Institut für Biochemie, Universität Leipzig, FRG.
Schellenberger
W
W
Kopperschläger
G
G
Hofmann
E
E
D000249:Adenosine Monophosphate / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004120:Dimethyl Suberimidate / Q000494:pharmacology*; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003432:Cross-Linking Reagents; D005635:Fructosediphosphates; D005636:Fructosephosphates; D046911:Macromolecular Substances; D004120:Dimethyl Suberimidate; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D010732:Phosphofructokinase-1