Mechanistic Implications from Crystalline Complexes of Wild-Type and Mutant Adenylosuccinate Synthetases from Escherichia coli

• Published in: Biochemistry (Easton) 1999-05, Vol.38 (21), p.6953-6961
• Main Authors: Choe, Jun-Yong, Poland, Bradley W, Fromm, Herbert J, Honzatko, Richard B
• Format: Article
• Language: English
• Subjects: adenylosuccinate synthase; Adenylosuccinate Synthase - chemistry; Adenylosuccinate Synthase - genetics; Adenylosuccinate Synthase - metabolism; Aspartic Acid - chemistry; Aspartic Acid - metabolism; Binding Sites - genetics; Crystallization; Crystallography, X-Ray; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Guanosine Diphosphate - chemistry; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; Hydrogen Bonding; Inosine Monophosphate - chemistry; Inosine Monophosphate - metabolism; Magnesium - chemistry; Magnesium - metabolism; Models, Molecular; Mutagenesis, Site-Directed
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990159s

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.