title: Characterization of intermediate species during the molecular assembly of aspartase.
abstract: Molecular assembly of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli was studied during the reversible denaturation. Although previous studies [Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochem. Biophys. Acta 480, 479-488] were unable to identify intermediate species during the course of reversible denaturation of aspartase, temperature-controlled HPLC and cross-linking with dimethyl suberimidate of the renaturation products showed that monomeric, dimeric and trimeric species occupied over 80% of the total oligomeric molecules below 13 degrees C; unlike the tetramer, these intermediates were without the activity. The degree of active tetramer formation was a linear function of the restoration of the activity below 18 degrees C, while above 23 degrees C, the activity regain was less than 70% restoration of tetrameric molecules. Upon examination by fluorescence spectroscopy, structural changes during reconstitution exhibited such complex kinetics that the rapid formation of structured oligomers proceeds first with a half-time of less than 10 sec, followed by slow subunit association. These results strongly suggest that the tetramer formation is an essential prerequisite, though not sufficient for the active enzyme.
authors.affiliation: Department of Chemistry, Faculty of Science, Kyoto University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Imaishi
authors.forename: N
authors.initials: N
authors.lastname: Yumoto
authors.forename: M
authors.initials: M
authors.lastname: Tokushige
mesh_terms: D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase