Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.
Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.
Choe
J Y
JY
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames 50011, USA.
Poland
B W
BW
Fromm
H J
HJ
Honzatko
R B
RB
D000265:Adenylosuccinate Synthase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007291:Inosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D007291:Inosine Monophosphate; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D006160:Guanosine Triphosphate; D000265:Adenylosuccinate Synthase; D008274:Magnesium