Entrapment of 6-thiophosphoryl-IMP in the active site of crystalline adenylosuccinate synthetase from Escherichia coli.
Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with Mg2+, 6-thiophosphoryl-IMP, GDP, and hadacidin at 298 and 100 K have been refined to R-factors of 0.171 and 0.206 against data to 2.8 and 2.5 A resolution, respectively. Interactions of GDP, Mg2+ and hadacidin are similar to those observed for the same ligands in the complex of IMP, GDP, NO3-, Mg2+ and hadacidin (Poland, B. W., Fromm, H. J. & Honzatko, R. B. (1996). J. Mol. Biol. 264, 1013-1027). Although crystals were grown from solutions containing 6-mercapto-IMP and GTP, the electron density at the active site is consistent with 6-thiophosphoryl-IMP and GDP. Asp-13 and Gln-224 probably work in concert to stabilize the 6-thioanion of 6-mercapto-IMP, which in turn is the nucleophile in the displacement of GDP from the gamma-phosphate of GTP. Once formed, 6-thiophosphoryl-IMP is stable in the active site of the enzyme under the conditions of the structural investigation. The direct observation of 6-thiophosphoryl-IMP in the active site is consistent with the putative generation of 6-phosphoryl-IMP along the reaction pathway of the synthetase.
Poland
B W
BW
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Bruns
C
C
Fromm
H J
HJ
Honzatko
R B
RB
D000265:Adenylosuccinate Synthase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D007291:Inosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data
C106606:6-thiophosphoryl-inosine monophosphate; D008024:Ligands; D007291:Inosine Monophosphate; D000265:Adenylosuccinate Synthase