Abstract
The kinetic mechanism of the phosphorylase kinase reaction was studied using a synthetic tetradecapeptide and phosphorylase b as substrates. The synthetic peptide contained the amino acid sequence, Se+Asp-Gln-Glu-LysArg1”-Lys-Gln-Ile-Ser-Va115-Arg-Gly-Leu as found in rabbit muscle phosphorylase 6. From initial rate studies and from the use of substrate analogs as competitive inhibitors, it was determined that the initial rate data were consistent with the Random Ri Ri mechanism for the phosphorylase kinase reaction. Although the synthetic tetradecapeptide appeared to be a good substrate for phosphorylase kinase (V, was only one-half of that with phosphorylase b as substrate), the K, of the peptide was 75-fold larger than the K,, of phosphorylase b. Various peptide analogs of the synthetic substrate were used to study the effect of size, charge at the NH,-terminal and the COOH-terminal residue, and substitution of arginine-16 on the kinetic parameters of activated phosphorylase kinase. Deletion of the first 4 residues and charged end groups on the peptide affected the rate of phosphorylation. Replacement of arginine-16 with a conservative or nonconservative substitution affected both the K, and rate of phosphorylation. A cyanogen bromide fragment derived from and containing the NH,-terminal segment of phosphorylase was tested for its ability to serve as substrate for activated phosphorylase kinase. The K,,, for activated phosphorylase kinase was intermediate for that of tetradecapeptide and phosphorylase b, while the V,,, was lower than with tetradecapeptide as substrate.
Highlights
The kinetic mechanism of the phosphorylase kinase reaction was studied using a synthetic tetradecapeptide and phosphorylase b as substrates
The synthetic substrate, that the in other words, must mimic the natural substrate in the characteristic features of the kinase reaction. This was demonstrated with the synthetic tetradecapeptide as substrate (5, II), (i) the correct seryl residue, serine-14, was phosphorylated; (ii) the characteristic lag was observed in the rate of product formation with nonactivated phosphorylase kinase; (iii) the activity ratio of pH 6.8 to 8.2 was similar to that seen with phosphorylase b as substrate
By using the model system one avoids the many difficulties associated with studying the phosphorylase kinase reaction where the protein substrate, phosphorylase b, has binding sites for Mg?+ [26], ATP
Summary
The kinetic mechanism of the phosphorylase kinase reaction was studied using a synthetic tetradecapeptide and phosphorylase b as substrates. 6. From initial rate studies and from the use of substrate analogs as competitive inhibitors, it was determined that the initial rate data were consistent with the Random Ri Ri mechanism for the phosphorylase kinase reaction. Various peptide analogs of the synthetic substrate were used to study the effect of size, charge at the NH,-terminal and the COOH-terminal residue, and substitution of arginine-16 on the kinetic parameters of activated phosphorylase kinase. Deletion of the first 4 residues and charged end groups on the peptide affected the rate of phosphorylation. Replacement of arginine-16 with a conservative or nonconservative substitution affected both the K, and rate of phosphorylation. The K,,, for activated phosphorylase kinase was intermediate for that of tetradecapeptide and phosphorylase b, while the V,,, was lower than with tetradecapeptide as substrate
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