Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine. |
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Authors: | T E Mansour R F Colman |
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Affiliation: | 1. Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305 USA;2. Department of Chemistry, University of Delaware, Newark, Delaware 19711 USA |
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Abstract: | The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO2BZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites. |
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