Tubulin-tyrosine ligase catalyzes covalent binding of 3-fluoro-tyrosine to tubulin: kinetic and [19F]NMR studies.
The use of 3-fluoro-tyrosine as an alternative substrate for the enzyme tubulin:tyrosine ligase which catalyzes the incorporation of tyrosine into the alpha-tubulin subunit was investigated. The incorporation of tyrosine into tubulin was inhibited competitively by 3-fluoro-tyrosine with an apparent Ki of approximately 25 microM. The affinity for this analog was similar to that of tyrosine, confirming that the hydrogen at position 3 of the aromatic ring is not essential for the reaction catalyzed by TTLase. The incorporation of 3-fluoro-tyrosine into the C-terminus of the alpha-tubulin subunit was demonstrated through [19F]NMR spectroscopy. The 3-fluoro-tyrosine signal at -58.6 ppm (trifluoroacetic acid as external standard), with a bandwidth of 24.7 Hz presented a chemical shift of 0.75 ppm upfield and an enlargement in the bandwidth (30.5 Hz) when incorporated into tubulin. These results strongly suggest that this amino acid is exposed to the solvent in tubulin. Tubulin covalently labeled with 3-fluoro-tyrosine was competent to polymerize into microtubules. The use of fluorinated tubulin in [19F]NMR spectroscopy for studying questions concerning protein conformation and interactions will be discussed.
Monasterio
O
O
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Nova
E
E
López-Brauet
A
A
Lagos
R
R
D000818:Animals; D002384:Catalysis; D002645:Chickens; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008870:Microtubules / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D014404:Tubulin; C001245:3-fluorotyrosine; D014443:Tyrosine; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase