Interaction of phosphorylase kinase with the 2',3'-dialdehyde derivative of adenosine triphosphate. 1. Kinetics of inactivation.

The 2',3'-dialdehyde derivative of ATP (oATP) was found to be a valid affinity label for rabbit skeletal muscle phosphorylase kinase. Inactivation by oATP at pH 6.8 followed pseudo-first-order and saturation kinetics. An apparent Ki of approximately 6.7 microM was obtained in the presence of 0.6 mM Ca2+ plus 10 mM Mg2+. Protection against the rate of inactivation was provided by the natural substrate ATP. In addition, at pH 8.2, oATP could be used as a substrate to phosphorylate phosphorylase b, thus providing evidence that oATP can bind to the active site of phosphorylase kinase. Inactivation of phosphorylase kinase by oATP was sensitive to various effectors of the enzyme such as Ca2+, Mg2+, and pH. Ca2+ plus Mg2+ synergistically enhanced the rate of inactivation severalfold; each metal ion by itself had little effect on the rate of inactivation. This synergism was seen both at pH 6.8 and at pH 8.2; however, the rates of inactivation were much greater at pH 6.8. The enhancement of inactivation by Ca2+ plus Mg2+ was also more pronounced with activated than with nonactivated kinase.