Phosphorylation of Sepharose-Coupled Peptides by Protein Kinase A

The kinetics of phosphorylation of the Sepharose-coupled peptide RRASVA by catalytic subunit of protein kinase A, and diastereomers of this peptide, containingd-amino acids successively in each position, were studied. Coupling of these peptides with the amino and carboxyl termini to CH- and AH-Sepharoses had similar effects on the phosphorylation reaction, increasing theKmand decreasing theVvalues, respectively. The diastereomeric peptides were also phosphorylated by the enzyme and the rate of this reaction depended on the position of substitution ofl-amino acids with theird-analogs. However, this dependence was much less pronounced if compared with stereoselectivity of the enzyme in reactions with these peptides in solution: theKmvalues for the Sepharose-coupled peptides were almost insensitive to the replacement ofl-amino acids withd-analogs and moderate stereoselectivity was revealed in the maximal velocity of the reaction. The Sepharose-coupled peptide containingd-serine was also phosphorylated by protein kinase A while the same peptide in solution did not interact with the enzyme. Consequently, the polymer, enveloping the phosphorylatable peptide, may remarkably influence the recognition of the reaction site, altering bothVandKmvalues.