Abstract
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.
Highlights
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)
A truncated form of the EGF receptor tyrosine kinase domain (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
We have investigated the potential of the fluorescent nucleotide analog 2Ј(3Ј)-O-(2,4,6-trinitrophenyl)adenosine 5Ј-triphosphate (TNP-ATP)1 as a molecular probe for enzymological studies of protein-tyrosine kinases
Summary
Materials—2Ј(3Ј)-O-(2,4,6-trinitrophenyl)adenosine 5Ј-triphosphate (TNP-ATP) was obtained from Molecular Probes, Inc. The affinity of the interaction of TNP-ATP with each of the TKD proteins was determined by titrating a fixed quantity of the TKD protein with increasing concentrations of the fluorescent nucleotide, while recording the fluorescence intensity at the wavelength maximum of the emission spectrum of enzyme-bound TNP-ATP (540 nm) with excitation at 418 nm. Where E0 and T0 are the total concentrations of added TKD protein and TNP-ATP nucleotide, respectively, KTNP-ATP is the dissociation constant characterizing the interaction, and ⌬F0 is the difference in the intrinsic fluorescence yields of enzyme-bound and free TNP-ATP. This equation was fit to the titration data with a nonlinear least squares algorithm and with KTNP-ATP, E0, and ⌬F0 as adjustable parameters. KЈTNP-ATP was determined at varying concentrations of S, and Equation 2 was fit to these data with KTNP-ATP, KS, and ␣ as adjustable parameters (see Fig. 7)
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