Nucleoside triphosphate specificity of tubulin.
We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.
Chakrabarti
G
G
Department of Molecular Biosciences and The NMR Laboratory, University of Kansas, Lawrence 66045-2106, USA.
Mejillano
M R
MR
Park
Y H
YH
Vander Velde
D G
DG
Himes
R H
RH
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007293:Inosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011685:Purine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D011685:Purine Nucleotides; D012265:Ribonucleotides; D014404:Tubulin; D007293:Inosine Triphosphate; C065326:xanthosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate