Location of the aromatic binding site and preparation of an aromatic derivative of glycogen phosphorylase.
Rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) was inactivated by 1,5-difluoro-2,4-dinitrobenzene at pH 7.6 at a rate much faster than by 1-fluoro-2,4-dinitrobenzene. The reaction was very specific at low concentration of 1,5-difluoro-2,4-dinitrobenzene. Glucose 1-phosphate, glucose 6-phosphate, AMP and ATP afforded some protection against inactivation by 1,5-difluoro-2,4-dinitrobenzene. These results and kinetic analyses of the modified enzyme were used to locate the binding site for aromatic compounds in phosphorylase. The above ligands and aromatic compounds are shown to bind on the enzyme in the same region which is located near the monomer/monomer interface. An apparently homogeneous dinitrodiphenyzene derivative of phosphorylase b with only one group per dimeric enzyme and having 50% of the catalytic activity was prepared. This derivative in which the subunits were not cross-linked by the reagent was devoid of the homotropic cooperativity for the substrate or activator sites even in the presence of allosteric inhibitors. Glucose behaved quite differently from other ligands in its effect on modification and on the kinetics of the modified enzyme. The significance of the glucose site is discussed.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology; D005958:Glucosephosphates / Q000494:pharmacology; D009124:Muscle Proteins / Q000302:isolation & purification; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011817:Rabbits
D005958:Glucosephosphates; D009124:Muscle Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004139:Dinitrofluorobenzene; D006005:Phosphorylases; C002041:1,5-difluoro-2,4-dinitrobenzene