title: Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue.
abstract: Two residues, K89 and S380, thought to interact with the gamma-carboxyl group of the substrate L-glutamate, have been altered by site-directed mutagenesis of clostridial glutamate dehydrogenase (GDH). The single mutants K89L and S380V and the combined double mutant K89L/S380V were constructed. All three mutants were satisfactorily overproduced in soluble form. However, only the K89L mutant was retained by the dye column normally used in purifying the wild-type enzyme. All three mutant enzymes were purified to homogeneity and tested for substrate specificity with 24 amino acids. The single mutant S380V showed no detectable activity. The alternative single mutant K89L showed an activity towards L-glutamate that was decreased nearly 2000-fold compared with wild-type enzyme, whereas the activities towards the monocarboxylic substrates alpha-aminobutyrate and norvaline were increased 2- to 3-fold. A similar level of activity was obtained with methionine (0.005 U/mg) and norleucine (0.012 U/mg), neither of which give any activity with the wild-type enzyme under the same conditions. The double mutant showed decreased activity with all substrates compared with the wild-type GDH. In view of its novel activities, the K89L mutant was investigated in greater detail. A strictly linear relationship between reaction velocity and substrate concentration was observed up to 80 mM L-methionine and 200 mM L-norleucine, implying very high Km values. Values of kcat/Km for L-methionine and L-norleucine were 6.7 x 10(-2) and 0.15 s-1 M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, Sheffield University, Western Bank, UK.
authors.forename: X G
authors.initials: XG
authors.lastname: Wang
authors.forename: K L
authors.initials: KL
authors.lastname: Britton
authors.forename: P J
authors.initials: PJ
authors.lastname: Baker
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
authors.forename: P C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D004926:Escherichia coli; D015870:Gene Expression; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D008239:Lysine