Modification and identification of glutamate residues at the arginine-recognition site in the catalytic subunit of adenosine 3' :5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle.
It has been proposed that the active centre of cyclic AMP-dependent protein kinase contains an arginine-recognition site, which is considered to be essential for the function of the catalytic subunit of the kinase [Matsuo, Huang & Huang (1978) Biochem. J.173, 441-447]. The catalytic subunit can be inactivated by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide and glycine ethyl ester at pH6.5. The enzyme can be protected from inactivation by preincubation with histone, a protein substrate of the enzyme. On the other hand, ATP, which also serves as a protein kinase substrate, does not afford protection. Polyarginine, a competitive inhibitor of protein kinase, which is known from kinetic studies to interact specifically with the arginine-recognition site, partially protects the catalytic subunit from inactivation by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide. These results lead to the conclusion that the site of modification by carbodi-imide/glycine ethyl ester is most likely located at the arginine-recognition site of the active centre. A value of 1.7+/-0.2 (mean+/-s.d.) mol of carboxy groups per mol of catalytic subunit has been obtained for the number of essential carboxy groups for the function of protein kinase; a complete chemical modification of these essential carboxy groups results in total loss of catalytic activity. Finally, we have identified the essential carboxy group in the catalytic subunit of cyclic AMP-dependent protein kinase as being derived from glutamate residues. This is achieved by a three-step procedure involving an extensive proteolytic digestion of the [1-(14)C]glycine ethyl ester-modified enzyme and two successive high-voltage electrophoreses of the hydrolysate. It is concluded that 1.7mol of glutamyl carboxy groups per mol of catalytic subunit may be considered a component of the arginine-recognition site in the active centre of cyclic AMP-dependent protein kinase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005971:Glutamates / Q000032:analysis; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006657:Histones / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D011817:Rabbits
D000596:Amino Acids; D005971:Glutamates; D006657:Histones; D010455:Peptides; D047428:Protein Kinase Inhibitors; C015462:polyarginine; C022010:glycine ethyl ester; D001120:Arginine; D000242:Cyclic AMP; D011494:Protein Kinases; D005022:Ethyldimethylaminopropyl Carbodiimide; D005998:Glycine