P. Cook, M. Neville, K. Vrana
Nov 9, 1982
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Journal
Biochemistry
Abstract
The kinetic mechanism for adenosine cyclic 3',5'-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg2+ to a second site. Increasing the Mg2+ results in a 5-6-fold decrease in V,,, in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/KMgATP in the absence of KCl and a 13-fold increase in V/KMdTP at 0.1 M KC1. The effect of increasing free Mg2+ on V,,, and V/K was also obtained with MgITP (20% the V,,, with MgATP) and MgGTP (10% the V,,, with MgATP) as substrates. The dissociation constant for Mg2+ from E-Ser-peptide-Mg2+ and central complexes is 2-3 mM. At low concentrations of free Adenosine cyclic 3',5'-monophosphate dependent protein kinase catalyzes the phorphorylation of a variety of proteins according to