Kinetic analysis of cAMP-dependent protein kinase: Mutations at histidine 87 affect peptide binding and pH dependence

Mutation of His87 in the catalytic (C-) subunit of the cAMP-dependent protein kinase (cAPK) led to changes in the kinetic properties of this enzyme. The C-subunit is a bilobal structure, with catalysis occurring in the cleft between the two lobes. His87 lies at the edge of the cleft, making an interaction with phosphothreonine, 197. This is the only direct electrostatic or hydrogen-bonding interaction between the small and large lobes. Solvent viscosity studies of the His87Ala mutant of the C-subunit (rC[H87A]) revealed that binding of two peptides, LRRASLG and LRRASLG-NH2 was impaired relative to that of the wild-type C-subunit. Consistent with this, the K-i's for two inhibitor peptides, LRRALG and LRRAALG-NH2, were 4 and 1.4 mM, respectively, 5- and 7-fold higher than the K-i's of the respective peptides for wild-type protein. Kinetic constants for three octapeptide substrates that differed only at the P+2 position suggested a direct interaction of His87 with residues at this site. The k(cat) for rC[H87A] was 2-3-fold higher than k(cat) for the wild-type enzyme, indicating an effect of the mutation on the rate-limiting step, product release. The pH dependence of kinetic parameters for rC[H87A] was also measured. A single pK(a) of 6.5 was observed in k(cat)/K-peptide as compared to the two pK(a)'s of 6.5 and 8.5 for the wild-type enzyme. These changes suggest a role for His87 in substrate recognition and in stabilization of the catalytically competent conformation of the enzyme.