The ATP-binding site in gamma subunit of phosphorylase kinase.
To reveal the structure of the ATP-binding site(s) in rabbit muscle phosphorylase kinase, we modified the enzyme with adenosine polyphosphopyridoxals. Adenosine tri- and tetraphosphopyridoxals at micromolar concentrations effectively inactivated the enzyme in a time-dependent manner. Inactivation of the enzyme was accelerated by the addition of Ca2+ and Mg2+. Protection from inactivation was afforded by adenylyl beta,gamma-imidodiphosphate and ADP. In reversible inhibition kinetics, adenosine polyphosphopyridoxals as well as their reduced compounds (adenosine polyphosphopyridoxines) competed with ATP. These results suggest that adenosine polyphosphopyridoxals bind to the ATP-binding site(s) in phosphorylase kinase. When phosphorylase kinase was incubated with adenosine triphosphopyridoxal in the presence of Ca2+ and Mg2+, incorporation of the label into alpha, beta, and gamma subunits was observed. In the absence of both cations, larger amounts of the label were incorporated into all the subunits. Structural study on adenosine triphosphopyridoxal-modified sites in the gamma subunit (having a catalytic site) revealed that Lys-151 is mainly labeled. Based on the results of the present and other studies, it is suggested that the site around Lys-151 is involved in recognition of the substrate protein.
Tagaya
M
M
Institute of Scientific and Industrial Research, Osaka University, Japan.
Hayakawa
Y
Y
Fukui
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011817:Rabbits
D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase