Stimulation of cGMP-dependent protein kinase I alpha by a peptide from its own sequence. An investigation by enzymology, circular dichroism and 1H NMR of the activity and structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide.
The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)
Huggins
J P
JP
Marion Merrell Dow Research Institute, Strasbourg, France.
Ganzhorn
A J
AJ
Saudek
V
V
Pelton
J T
JT
Atkinson
R A
RA
D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000494:pharmacology; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006152:Cyclic GMP / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000008:administration & dosage / Q000494:pharmacology*
D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP