Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.
Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.
Glass
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DB
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
Feller
M J
MJ
Levin
L R
LR
Walsh
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D047428:Protein Kinase Inhibitors; C061479:protein kinase inhibitor peptide; C037642:kemptide; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP