Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli

Biochemistry. 1999 May 25;38(21):6953-61. doi: 10.1021/bi990159s.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylosuccinate Synthase / chemistry*
  • Adenylosuccinate Synthase / genetics*
  • Adenylosuccinate Synthase / metabolism
  • Aspartic Acid / chemistry
  • Aspartic Acid / metabolism
  • Binding Sites / genetics
  • Crystallization
  • Crystallography, X-Ray
  • 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*

Substances

  • Inosine Monophosphate
  • Guanosine Diphosphate
  • Aspartic Acid
  • Guanosine Triphosphate
  • Adenylosuccinate Synthase
  • Magnesium

Associated data

  • PDB/1CG0
  • PDB/1CG1
  • PDB/1CG3
  • PDB/1CG4