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 occurrin g in the cleft between the two lobes. His87 lies at the edge of the cl eft, making an interaction with phosphothreonine, 197. This is the onl y direct electrostatic or hydrogen-bonding interaction between the sma ll and large lobes. Solvent viscosity studies of the His87Ala mutant o f the C-subunit (rC[H87A]) revealed that binding of two peptides, LRRA SLG and LRRASLG-NH2 was impaired relative to that of the wild-type C-s ubunit. Consistent with this, the K-i's for two inhibitor peptides, LR RALG and LRRAALG-NH2, were 4 and 1.4 mM, respectively, 5- and 7-fold h igher 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 resi dues 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 com petent conformation of the enzyme.