Holoenzyme interaction sites in the cAMP-dependent protein kinase. Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit.
Two mutations of the catalytic (C) subunit of the cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli and purified. These mutants were phosphorylated at Thr197 and were catalytically active, although some changes in their kinetic parameters were observed. The most striking differences were in their interaction with the physiological inhibitors. Both mutants were inhibited by protein kinase inhibitor with Ki values below 50 nM. Both mutants were defective in their interaction with the type I regulatory (RI) subunit as measured by (i) the rate of holoenzyme formation with cAMP bound RI-subunit and (ii) the apparent Kd with the cAMP-free RI-subunit. The rate of holoenzyme formation was impaired both in the presence and absence of ATP with the His to Asp mutant showing the greatest effect. The mutant C-subunits were also combined with RI-subunits that contained mutations in the autoinhibitor sequence at Arg94 (P-3) and Ser99 (P + 2). Complementarity between His87 and Ser99 was established, but not between His87 and Arg94. Holoenzyme formation with a Ser99-->Lys mutant RI-subunit was less dependent on ATP when combined with either of the C-subunit mutants than when it was combined with the wild-type C-subunit. The apparent Kd values in the presence of ATP for the mutant combinations were also measured. The Ser99-->Lys mutant was compensated for by both His87 mutants. The His87-->Ala C-subunit mutant was unable to form an inhibited holoenzyme complex with a mutant RI-subunit which was defective in cAMP binding to the A-site. This indicated that this R-subunit was defective in C-subunit recognition as well as in cAMP binding. The roles of His87 on the C-subunit and Ser99 and Arg209 on the RI-subunit in R-C interactions are discussed.
Cox
S
S
Department of Chemistry, University of California, San Diego, La Jolla 92093-0654.
Taylor
S S
SS
D000409:Alanine; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine