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Abstract: 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 K(m) 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