Bai Ruoli, Vanderwal Christopher D, Díaz J Fernando, Hamel Ernest
Toxicology and Pharmacology Branch, Developmental Therapeutics Program, National Cancer Institute, National Institutes of Health,Frederick, MD 21702, USA.
J Nat Prod. 2008 Mar;71(3):370-4. doi: 10.1021/np800056m. Epub 2008 Feb 26.
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.
天然产物环链菌素与微管蛋白以共价和化学计量的方式发生反应，作用于β-微管蛋白的两个氨基酸残基之一，即苏氨酸-218或天冬酰胺-226，但在未聚合的微管蛋白中反应程度低得多，且仅作用于苏氨酸-218。研究发现，8-乙酰基环链菌素（8AcCS）诱导微管蛋白组装的方式与环链菌素几乎相同。因此，我们合成了[(14)C-乙酰基]8AcCS，并研究了其与戊二醛稳定化微管以及未组装微管蛋白相互作用的动力学。对于微管，我们发现8AcCS迅速结合，在共价反应发生之前有一个极小的（用放射性标记类似物无法测量）延迟期。整个反应的表观反应速率常数范围从0℃时的6.2×10(2)M(-1)s(-1)到20℃时的5.6×10(3)M(-1)s(-1)。在0℃和10℃获得的速率常数表明该反应的活化能约为27千卡/摩尔，而在10℃和20℃获得的速率常数表明活化能约为7.7千卡/摩尔。对于未聚合的微管蛋白，我们确实发现发生了极小的共价反应但没有明显的微管组装，而只有在组装后才发生大量反应。总之，放射性标记的8AcCS表明配体与微管蛋白的广泛共价相互作用需要微管组装，并且共价反应在初始结合相互作用后迅速发生。
