A soluble insulin receptor kinase catalyzes ordered phosphorylation at multiple tyrosines of dodecapeptide substrates.

At present, the requirements for efficient phosphorylation of exogenous substrates by protein-tyrosine kinases are largely unknown. The proton resonances of each of the 3 tyrosines of the dodecapeptide substrate RRDIYETDYYRK are well resolved in the aromatic region of the 1H NMR spectra: thus, it is feasible to directly monitor phosphorylation at each site. A soluble approximately 48-kDa derivative of the human insulin receptor cytoplasmic protein-tyrosine kinase domain phosphorylates this peptide at all 3 tyrosine sites and does so in a highly ordered and progressive manner (Y9, then Y10 and finally Y5), proceeding to full stoichiometry at each site before phosphorylating the next. This experimental system now provides an approach by which to follow the stereochemical requirements and dynamics of substrate phosphorylation by a protein-tyrosine kinase in solution in real time.