Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology


The specificity of phosphorylase kinase and cAMP-dependent protein kinase was studied by means of peptide and protein substrate analogs of phosphorylase. A peptide, leu-ser-tyr-arg-arg-tyr-ser-leu, which has a predicted (beta)-turn sequence, was synthesized. cAMP-dependent protein kinase was able to phosphorylate this peptide on the C-terminal serine, and phosphorylase kinase on the N-terminal serine. This supports earlier theories that cAMP-dependent protein kinase recognizes substrates with arginines N-terminal to the serine, while phosphorylase kinase has an opposite polarity in arginine recognition. A D-peptide analog of the above peptide was also synthesized in hopes of reversing the polarity. It failed to be a substrate for either kinase but was an inhibitor for both;Earlier work has shown that peptide analogs of the phosphorylation site of phosphorylase b are substrates for both kinases, so we were interested in examining the role of the organized structure of phosphorylase in regulating the specificity. By CNBr-digestion, we isolated an N-terminal fragment of phosphorylase and determined that it retained some organized structure. This fragment was a substrate for phosphorylase kinase, with a K(,m) value lower than those of synthetic peptide substrates, but was a very poor substrate for cAMP-dependent protein kinase, showing that the specificity elements for the two kinases are contained in the N-terminal region. We also synthesized a peptide analog, sequence 9-24 of phosphorylase, as earlier peptides had been analogs of the 5-18 sequence. We showed that this peptide was a much better substrate for phosphorylase kinase, and a worse one for cAMP-dependent protein kinase, showing that the region C-terminal to the phosphorylated serine is especially important in regulating kinase specificity;We have begun studies of the chemical role of arginyl residues in kinase specificity, with model studies of the reactivity of p-nitrophenyl-glyoxal with arginine analogs. We have shown that the reagent's reactivity is related to the pK(,a) of the analog, and that the pKa's of the analog can be altered by environmental effects, such as being incorporated into a detergent micelle. Thus, we can study how the protonation state and the environment of an arginine residue can effect its role in specificity.



Digital Repository @ Iowa State University,

Copyright Owner

Michael Owen Hurst



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120 pages

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Biochemistry Commons