Kinetic analysis of protein kinase C: product and dead-end inhibition studies using ADP, poly(L-lysine), nonhydrolyzable ATP analogues, and diadenosine oligophosphates.
The kinetic mechanism of protein kinase C (PKC) was analyzed via inhibition studies using the product MgADP, the nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imidotriphosphate) (MgAMPPNP), the peptide antagonist poly(L-lysine), and several naturally occurring ATP analogues that are produced in rapidly growing cells, i.e., the diadenosine oligophosphates (general structure: ApnA; n = 2-5). By use of histone as the phosphate acceptor, the inhibition of PKC by MgAMPPNP and MgADP was found to be competitive vs MgATP (suggesting that these compounds bind to the same enzyme form), whereas their inhibition vs histone was observed to be noncompetitive. In contrast, the inhibition by poly(L-lysine) appeared competitive vs histone but uncompetitive vs MgATP, which is consistent with a model wherein MgATP binding promotes the binding of poly(L-lysine) or histone. With the diadenosine oligophosphates, the degree of PKC inhibition was found to increase according to the number of intervening phosphates. The diadenosine oligophosphates Ap4A and Ap5A were the most effective antagonists of PKC, with Ap5A being approximately as potent as MgADP and MgAMPPNP. However, as opposed to MgADP and MgAMPPNP, Ap4A and Ap5A appear to act as noncompetitive inhibitors vs both MgATP and histone, suggesting that they can interact at several points in the reaction pathway. These studies support the concept of a steady-state mechanism where MgATP binding preferentially precedes that of histone, followed by the release of phosphorylated substrate and MgADP. Furthermore, these results indicate a differential interaction of the diadenosine oligophosphates with PKC, when compared to other adenosine nucleotides.
Leventhal
P S
PS
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Bertics
P J
PJ
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D011107:Polylysine / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051381:Rats
D015226:Dinucleoside Phosphates; D011107:Polylysine; D000266:Adenylyl Imidodiphosphate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C020733:diadenosine tetraphosphate; D000244:Adenosine Diphosphate; D011493:Protein Kinase C