Nucleoside Triphosphate Specificity of Tubulin

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• 作者：Gopal Chakrabarti ; Marisan R. Mejillano ; Young-Hee Park ; David G. Vander Velde ; and Richard H. Himes
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 22, 2000
• 年：2000
• 卷：39
• 期：33
• 页码：10269 - 10274
• 全文大小：83K
• 年卷期：v.39,no.33(August 22, 2000)
• ISSN：1520-4995

文摘

We have determined the binding affinity for binding of the four purine nucleoside triphosphatesGTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. Therelative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclicamino group in GTP is important in determining the nucleotide specificity of tubulin and may interactwith a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to thehigh affinty for GTP. NMR ROESY experiments indicate that the four nucleotides have different averageconformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a mediumanti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformationcontributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, thereappears to be little difference in the ability of the four nucleotides to stimulate assembly. The criticalprotein concentration is essentially identical in reactions using the four nucleotides. All four of thenucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into themicrotubule. We also examined the binding of two ges/gifchars/gamma.gif" BORDER=0 >-phosphoryl-modified GTP photoaffinity analogues,p³-1,4-azidoanilido-GTP and p³-1,3-acetylanilido-GTP. These analogues are inhibitors of the assemblyreaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affintyfor GTP. The affinity of GTP is less sensitive to substitutions at the ges/gifchars/gamma.gif" BORDER=0 >-phosphoryl position that to changesin the purine ring.