title: Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli.
abstract: The high-resolution structure of l-aspartate ammonia-lyase from Escherichia coli has recently been determined [Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., & Farber, G. K. (1997) Biochemistry 36, 9136-9144]. An examination of the putative active site has been carried out, with the active site located in a cleft that contains the functionally significant lysine 327. A list of potential active site residues has been generated based on their proximity to this active site lysine, sequence homology comparisons with other members of the aspartase-fumarase enzyme family, and the necessity for chemically reasonable functionalities for the proposed roles. The five most likely candidates in the putative active site cleft have been examined by site-directed mutagenesis to test their feasibility for either substrate binding or acid-base catalytic roles. Arginine and lysine residues have been identified that appear to function in the orientation and binding of aspartic acid at the enzyme active site. Some tentative assignments have also been made of the acid and base catalytic groups that are proposed to be involved in the deamination reaction.
authors.affiliation: Department of Chemistry, University of Akron, Ohio 44325, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Jayasekera
authors.forename: W
authors.initials: W
authors.lastname: Shi
authors.forename: G K
authors.initials: GK
authors.lastname: Farber
authors.forename: R E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D001220:Aspartate Ammonia-Lyase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001220:Aspartate Ammonia-Lyase