Kinetics of the phosphotransferase reaction of the catalytic subunit of the tick salivary gland cAMP-dependent protein kinase

Abstract

The catalytic subunit of the cAMP dependent protein kinase was purified 100-fold from tick salivary glands. The enzyme mechanism of the phosphotransferase reaction catalyzed by this subunit was investigated. Highly purified enzyme did not show ATP-ase activity in the absence of protein substrates. Initial velocities were measured using histone H1 or a synthetic heptapeptide, Kemptide, as P i acceptors and [γ- 32P]ATP as a phosphodonor. At varying levels of ATP and fixed concentrations of protein cosubstrates and vice versa, double reciprocal plots gave a family of lines converging to the left of the 1 v axis. Secondary plots of intercepts and slopes versus fixed cosubstrate concentrations were linear. At high concentration (2 Km), histone showed substrate inhibition which was noncompetitive versus ATP. Product inhibition by Mg·ADP was competitive versus ATP and noncompetitive with respect to H1. Phosphohistone on the other hand was noncompetitive with respect to H1, but gave parabolic competitive inhibition against ATP. Dead end inhibition by AMP-PNP, an analogue of ATP, was competitive and noncompetitive against ATP and H1, respectively. Dead end inhibition by the protein kinase inhibitor was noncompetitive and competitive against ATP and H1, respectively. These studies strongly suggest that the tick salivary gland protein kinase has a sequential mechanism with predominately ordered addition of ATP followed by protein substrate and ordered release of phosphoprotein and ADP.