Interaction of a cyclostreptin analogue with the microtubule taxoid site: the covalent reaction rapidly follows binding.
The natural product cyclostreptin reacts covalently and stoichiometrically with microtubules, at either of two amino acid residues of beta-tubulin, Thr-218 or Asn-226, but much less extensively and only at Thr-218 in unpolymerized tubulin. It was found that 8-acetylcyclostreptin (8AcCS) induces tubulin assembly in a manner almost identical with that of cyclostreptin. We therefore synthesized [ (14)C-acetyl]8AcCS and studied the kinetics of its interaction with glutaraldehyde-stabilized microtubules and with unassembled tubulin. With the microtubules, we found that 8AcCS bound rapidly, with a minimal (unmeasurable with the radiolabeled analogue) lag prior to the occurrence of the covalent reaction. Apparent reaction rate constants for the overall reaction ranged from 6.2 x 10 (2) M (-1) s (-1) at 0 degrees C to 5.6 x 10 (3) M (-1) s (-1) at 20 degrees C. The rate constants obtained at 0 and 10 degrees C indicate an activation energy for the reaction of about 27 kcal/mol, while those obtained at 10 and 20 degrees C indicate an activation energy of about 7.7 kcal/mol. With the unpolymerized tubulin, we did find a minimal covalent reaction occurred without apparent microtubule assembly, but a substantial reaction only occurred following assembly. In conclusion, the radiolabeled 8AcCS shows that an extensive covalent interaction of ligand with tubulin requires microtubule assembly and that the covalent reaction occurs rapidly after the initial binding interaction.
Bai
Ruoli
R
Toxicology and Pharmacology Branch, Developmental Therapeutics Program, National Cancer Institute, National Institutes of Health,Frederick, MD 21702, USA.
Vanderwal
Christopher D
CD
Díaz
J Fernando
JF
Hamel
Ernest
E
D001216:Asparagine / Q000737:chemistry; D006576:Heterocyclic Compounds, 4 or More Rings / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism*; D015394:Molecular Structure; D011083:Polycyclic Compounds / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D043823:Taxoids / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D050257:Tubulin Modulators / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*
C528880:8-acetylcyclostreptin; C408303:FR 182877; D006576:Heterocyclic Compounds, 4 or More Rings; D011083:Polycyclic Compounds; D043823:Taxoids; D014404:Tubulin; D050257:Tubulin Modulators; D013912:Threonine; D001216:Asparagine