The overall kinetic mechanism of cyclic AMP-dependent protein kinase from bovine heart will be determined using a variety of different techniques. Initial velocity studies in the absence of inhibitors and in the presence of product and dead-end inhibitors will be carried out in the direction of phosphorylation of MgADP. Since results obtained from these studies are rarely conclusive, the above experiments will be complemented using isotope exchange at equilibrium for the MgATP/MgADP, MgATP/ phosphopeptide and peptide/phosphopeptide exchange reactions. Isotope partitioning experiments with 3H-peptide, 3H-ADP and 32P-phosphopeptide will also be used to corroborate kinetic mechanism. Finally, isotope exchange of beta-nonbridged 180-ADP into the bridge position, if it occurs will be carried out as a function of phosphopeptide to determine whether release of phosphopeptide and MgADP is random. Information on the location and amount of limitation of rate determining steps will Be determined from all of the above experimental approaches with additional information obtained from isotope effect studies. Information on the chemical mechanism will be obtained by completing the pH studies in the direction of MgADP phosphorylation to elucidate acid-based chemistry and through the use of heavy atom isotope effects using the remote label technique. Peptides containing beta-180-serine (depleted of 160) and 15N-glycine depleted of 14N and beta-160-serine (depleted of 180) and 14N- glycine (depleted of 15N will be prepared, mixed to the natural abundance of the glycine nitrogen and used to obtain the 180 isotope effect with 15N as the reporter. A number of alternate peptide substrates, nucleotides and conditions will be tested to find conditions for rate limiting phosphoryl transfer.
Showing the most recent 10 out of 12 publications
