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

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase k inase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous ro le to the phosphorylatable residues in other protein kinases is Glu182, whi ch makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the acti vation loop to the catalytic machinery. The double mutant R148A/E182S has b een kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the k(cat)/K-m 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 ha s a major role in substrate binding but also in correct orientation of the groups participating in catalysis.