title: Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase.
abstract: Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates. Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding. Asp-66 is a new residue found to be important in enzyme activity. The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntn-amidohydrolase family of enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama 36688, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: C
authors.initials: C
authors.lastname: Guan
authors.forename: N N
authors.initials: NN
authors.lastname: Aronson
mesh_terms: D019943:Amino Acid Substitution; D001227:Aspartylglucosylaminase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005417:Flavobacterium / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001227:Aspartylglucosylaminase