Determinants of l-Aspartate and IMP Recognition inEscherichia coli Adenylosuccinate Synthetase

• Published in: The Journal of biological chemistry Vol. 277; no. 11; pp. 8817 - 8821
• Main Authors: Gorrell, Andrea, Wang, Wenyan, Underbakke, Eric, Hou, Zhenglin, Honzatko, Richard B., Fromm, Herbert J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15.03.2002; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111810200

Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase fromEscherichia coli reveal significant roles for Val273 and Thr300 in the recognition ofl-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr300 to alanine increases the Km forl-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the Km forl-aspartate, while increasing kcatby 3-fold. Mutations of Val273 to alanine, threonine, or asparagine increase the Km forl-aspartate from 15- to 40-fold, and concomitantly decrease the Ki for dicarboxylate analogues ofl-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 Thr128 and Thr129 increase theKm for IMP by up to 30-fold and the alanine mutation of Thr301 abolishes catalysis supported byl-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented.