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Title
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Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).
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Authors
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D.Bossemeyer,
R.A.Engh,
V.Kinzel,
H.Ponstingl,
R.Huber.
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Ref.
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Embo J, 1993,
12,
849-859.
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PubMed id
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Abstract
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The crystal structure of the porcine heart catalytic subunit of cAMP-dependent
protein kinase in a ternary complex with the MgATP analogue MnAMP-PNP and a
pseudosubstrate inhibitor peptide, PKI(5-24), has been solved at 2.0 A
resolution from monoclinic crystals of the catalytic subunit isoform CA. The
refinement is presently at an R factor of 0.194 and the active site of the
molecule is well defined. The glycine-rich phosphate anchor of the nucleotide
binding fold motif of the protein kinase is a beta ribbon acting as a flap with
conformational flexibility over the triphosphate group. The glycines seem to be
conserved to avoid steric clash with ATP. The known synergistic effects of
substrate binding can be explained by hydrogen bonds present only in the ternary
complex. Implications for the kinetic scheme of binding order are discussed. The
structure is assumed to represent a phosphotransfer competent conformation. The
invariant conserved residue Asp166 is proposed to be the catalytic base and
Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is
functionally replaced by an Arg displaced by two residues in the primary
sequence, suggesting invariance in three-dimensional space. The structure
supports an in-line transfer with a pentacoordinate transition state at the
phosphorus with very few nuclear movements.
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