The kinetics of tyrosine phosphorylation by the purified epidermal growth factor receptor kinase of A-431 cells.
The protein kinase associated with the purified epidermal growth factor (EGF) receptor from membrane (Mr = 150,000) or vesicle (Mr = 170,000) preparations of A-431 cells was shown to catalyze the phosphorylation of the peptide Leu-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly at the tyrosine residue. EGF enhanced peptide phosphorylation by 3-5-fold. The steady state kinetic analysis of the purified kinase from membranes showed that the reaction mechanism was of the sequential type in either the presence of absence of EGF. Thus, the peptide and ATP must bind to the enzyme before any product is released. Both neurotensin 8-13 and kyotorphin were inhibitors but not substrates of the protein kinase. Kyotorphin was a linear noncompetitive inhibitor with ATP as the variable substrate and a linear competitive inhibitor with peptide as the variable substrate. ADP, a product of the kinase reaction, was a linear noncompetitive inhibitor with respect to ATP and a linear competitive inhibitor with respect to peptide. Based on these data, it can be suggested that the tyrosine protein kinase from A-431 cells catalyzes a Ordered Bi Bi reaction where peptide is the first substrate to bind and ADP is the last product to be released.
D002294:Carcinoma, Squamous Cell / Q000201:enzymology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D066246:ErbB Receptors; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification; D014443:Tyrosine
D011956:Receptors, Cell Surface; D014443:Tyrosine; D011494:Protein Kinases; D066246:ErbB Receptors