PHOSPHORYL EXCHANGE IS INVOLVED IN THE MECHANISM OF THE INSULIN-RECEPTOR KINASE

The cytoplasmic kinase domain of the human insulin receptor (IRKD; M(r) 49 kDa) has been over-expressed in insect cells using the baculovirus expression system. To investigate the kinase mechanism, we have compared the stoichiometry of ADP formation and phosphoryl transfer. After an initial phase of autophosphorylation, ATP is consumed without a stoichiometric increase in incorporated phosphate. During substrate phosphorylation using poly(Glu:Tyr) (4:1) phosphoryl transfer comes close to ATP turnover, which is independent of the presence of the substrate, indicating an increased efficiency (i.e. ATP turnover/phosphate incorporation) of phosphoryl transfer. Autophosphorylation under pulse-chase conditions suggests the existence of a phosphoenzyme intermediate.