Kinetic characterization of the double mutant R148A/E182S of glycogen phosphorylase kinase catalytic subunit: the role of the activation loop

• Published in: Journal of Protein Chemistry 2000-08, Vol.19 (6), p.499-505
• Main Authors: Skamnaki, V T, Oikonomakos, N G
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Animals; Arginine - genetics; Catalysis; Enzyme Activation; Glutamic Acid - genetics; Glycogen; Glycogen phosphorylase; Glycogens; Kinases; Kinetics; Models, Molecular; Molecular biology; Mutants; Mutation; Phosphorylase; Phosphorylase kinase; Phosphorylase Kinase - chemistry; Phosphorylase Kinase - genetics; Phosphorylase Kinase - metabolism; Phosphorylases; Phosphorylation; Protein Conformation; Protein kinase; Proteins; Rabbits; Residues; Serine - genetics; Substrate Specificity; Substrates
• ISSN: 0277-8033; 1572-3887; 1573-4943
• DOI: 10.1023/A:1026553532289

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase kinase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous role to the phosphorylatable residues in other protein kinases is Glu182, which makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the activation loop to the catalytic machinery. The double mutant R148A/E182S has been kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the kcat/Km value of the enzyme. The kinetic data, discussed in the light of the structural data from previously determined complexes of the enzyme, lead to the suggestion that the activation loop has a major role in substrate binding but also in correct orientation of the groups participating in catalysis.