TAILIEUCHUNG - Báo cáo khoa học: Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1

Aspartate ammonia lyases (also referred to as aspartases) catalyze the revers-ible deamination of l-aspartate to yield fumarate and ammonia. In the pro-posed mechanism for these enzymes, an active site base abstracts a proton from C3 ofl-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the prod-uct, fumarate. | ễFEBS Journal Site-directed mutagenesis kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1 Vinod Puthan Veetil1 Hans Raj1 Wim J. Quax1 Dick B. Janssen2 and Gerrit J. Poelarends1 1 Department of PharmaceuticalBiology Groningen Research Institute of Pharmacy University of Groningen The Netherlands 2 Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen The Netherlands Keywords aspartase aspartate ammonia lyase Bacillus deamination enzyme mechanism Correspondence G. J. Poelarends Department of PharmaceuticalBiology Groningen Research Institute of Pharmacy University of Groningen Antonius Deusinglaan 1 9713 AV Groningen The Netherlands Fax 31 50 3633000 Tel 31 50 3633354 E-mail Received 23 January 2009 revised 6 March 2009 accepted 20 March 2009 doi Aspartate ammonia lyases also referred to as aspartases catalyze the reversible deamination of L-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes an active site base abstracts a proton from C3 of L-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product fumarate. Although two crystal structures of aspartases have been determined details of the catalytic mechanism have not yet been elucidated. In the present study eight active site residues Thr101 Ser140 Thr141 Asn142 Thr187 His188 Lys324 and Asn326 were mutated in the structurally characterized aspartase AspB from Bacillus sp. YM55-1. On the basis of a model of the complex in which L-aspartate was docked manually into the active site of AspB the residues responsible for binding the amino group of L-aspartate were predicted to be Thr101 Asn142 and His188. This postulate is supported by the mutagenesis studies mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in .

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