title: Determinants of L-aspartate and IMP recognition in Escherichia coli adenylosuccinate synthetase.
abstract: Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase from Escherichia coli reveal significant roles for Val(273) and Thr(300) in the recognition of l-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr(300) to alanine increases the K(m) for l-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the K(m) for l-aspartate, while increasing k(cat) by 3-fold. Mutations of Val(273) to alanine, threonine, or asparagine increase the K(m) for l-aspartate from 15- to 40-fold, and concomitantly decrease the K(i) for dicarboxylate analogues of l-aspartate by up to 40-fold. The above perturbations are comparable with those resulting from the elimination of a hydrogen bond between the enzyme and substrate: alanine mutations of Thr(128) and Thr(129) increase the K(m) for IMP by up to 30-fold and the alanine mutation of Thr(301) abolishes catalysis supported by l-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Gorrell
authors.forename: Wenyan
authors.initials: W
authors.lastname: Wang
authors.forename: Eric
authors.initials: E
authors.lastname: Underbakke
authors.forename: Zhenglin
authors.initials: Z
authors.lastname: Hou
authors.forename: Richard B
authors.initials: RB
authors.lastname: Honzatko
authors.forename: Herbert J
authors.initials: HJ
authors.lastname: Fromm
mesh_terms: D000265:Adenylosuccinate Synthase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007291:Inosine Monophosphate / Q000378:metabolism*; D007700:Kinetics
chemical_list: D007291:Inosine Monophosphate; D001224:Aspartic Acid; D000265:Adenylosuccinate Synthase