Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase.
A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D008024:Ligands; D011494:Protein Kinases