Structural Specificity of the Adenosine 5'-Phosphate Site on Glycogen Phosphorylase b

• Published in: The Journal of biological chemistry 1970-08, Vol.245 (16), p.4058-4066
• Main Authors: Mott, D M, Bieber, A L
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides; Adenosine Triphosphate; Animals; Binding Sites; Chemical Phenomena; Chemistry; Enzyme Activation; Fluorescence; Fluorometry; Glucosyltransferases - antagonists & inhibitors; Glycogen; Hexosephosphates; Hydrogen-Ion Concentration; Kinetics; Mathematics; Muscles - enzymology; Nucleosides; Nucleotides; Pentosephosphates; Protamines; Pyridoxal Phosphate; Rabbits
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)62885-6

Twenty-three structural analogues of AMP were tested for their ability to activate and bind phosphorylase b in the presence and absence of substrate or protamine sulfate (or both). Michaelis constants, maximal velocities, and inhibition constants were measured by nucleotide activation of phosphorylase b and compared with the corresponding dissociation and inhibition constants measured by nucleotide quenching of a pyridoxal 5'-phosphate-associated enzyme fluorescence. The excitation maximum was 347 mµ and the emission maximum was 522 mµ. A sigmoidal saturation curve was obtained for AMP quenching of enzyme fluorescence. This, coupled with the ATP inhibition and the glucose 1-phosphate enhancement of AMP quenching of enzyme fluorescence, identified the site of AMP binding for enzyme fluorescence quenching with the AMP binding site for activation of phosphorylase b . Protamine enhanced the binding of some nucleotides to phosphorylase b but apparently affected only the V max of others as measured by nucleotide activation of the enzyme. An increase in enzyme fluorescence associated with addition of ATP and a quenching effect by added AMP or glucose-1-P suggested that the observed quenching was the result of the protein conformational state changes associated with the allosteric properties of the enzyme. Nucleotide analogues of AMP exhibited different heterotropic binding properties with phosphorylase b . Binding studies in the presence and absence of substrate revealed that for some nucleotides the affinity was unchanged while others showed less affinity for enzyme in the presence of substrate. Enzyme activation by bound nucleotide was sensitive to the integrity of both the adenine ring and the 5'-phosphate, but the latter appeared to be the most critical structural feature. Both the 5'-phosphate and purine ring were involved in nucleotide binding.