PHOSPHORYLATION OF SEPHAROSE-COUPLED PEPTIDES BY PROTEIN-KINASE-A

The kinetics of phosphorylation of the Sepharose-coupled peptide RRASV A by catalytic subunit of protein kinase A, and diastereomers of this peptide, containing D-amino acids successively in each position, were studied. Coupling of these peptides with the amino and carboxyl termin i to CH- and AH-Sepharoses had similar effects on the phosphorylation reaction, increasing the K-m and decreasing the V values, respectively . The diastereomeric peptides were also phosphorylated by the enzyme a nd the rate of this reaction depended on the position of substitution of L-amino acids with their D-analogs. However, this dependence was mu ch less pronounced if compared with stereoselectivity of the enzyme in reactions with these peptides in solution: the K-m values for the Sep harose-coupled peptides were almost insensitive to the replacement of L-amino acids with D-analogs and moderate stereoselectivity was reveal ed in the maximal velocity of the reaction. The Sepharose-coupled pept ide containing D-serine was also phosphorylated by protein kinase A wh ile the same peptide in solution did not interact with the enzyme. Con sequently, the polymer, enveloping the phosphorylatable peptide, may r emarkably influence the recognition of the reaction site, altering bot h V and K-m values. (C) 1996 Academic Press, Inc.