Nucleotide binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe.
The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.
Department of Pharmacology, University of Iowa, College of Medicine, Iowa City 52242-1109, USA.
K
K
Cheng
J G
JG
Koland
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D015335:Molecular Probes; D009711:Nucleotides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D015335:Molecular Probes; D009711:Nucleotides; D010455:Peptides; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D066246:ErbB Receptors