title: Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases.
abstract: Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Cristina V
authors.initials: CV
authors.lastname: Iancu
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Tudor
authors.initials: T
authors.lastname: Borza
authors.forename: Herbert J
authors.initials: HJ
authors.lastname: Fromm
authors.forename: Richard B
authors.initials: RB
authors.lastname: Honzatko
mesh_terms: D000265:Adenylosuccinate Synthase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D003854:Deoxyribonucleotides / Q000378:metabolism; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D003854:Deoxyribonucleotides; D007291:Inosine Monophosphate; D000265:Adenylosuccinate Synthase