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Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase.
- Source :
-
Biochemistry [Biochemistry] 2005 Mar 01; Vol. 44 (8), pp. 2982-92. - Publication Year :
- 2005
-
Abstract
- The E. coli isozyme of gamma-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and alpha-keto acids. The roles of the active site residues V241, E211, and I50 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The beta-branched side chain of V241 facilitates formation of external aldimine intermediates with primary amine substrates, while E211 provides charge compensation of R398 selectively in the primary amine half-reaction and I50 forms a hydrophobic lid at the top of the substrate binding site. The structures of the I50Q, V241A, and E211S mutants were solved by X-ray crystallography to resolutions of 2.1, 2.5, and 2.52 A, respectively. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle. Therefore, an attempt was made to convert GABA-AT into a decarboxylation-dependent aminotransferase similar to dialkylglycine decarboxylase by systematic mutation of E. coli GABA-AT active site residues. Two of the twelve mutants presented, E211S/I50G/C77K and E211S/I50H/V80D, have approximately 10-fold higher decarboxylation activities than the wild-type enzyme, and the E211S/I50H/V80D has formally changed the reaction specificity to that of a decarboxylase.
- Subjects :
- Amino Acid Substitution
Binding, Competitive
Crystallography, X-Ray
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Protein Conformation
Recombinant Proteins chemistry
Recombinant Proteins metabolism
gamma-Aminobutyric Acid metabolism
4-Aminobutyrate Transaminase chemistry
4-Aminobutyrate Transaminase metabolism
Escherichia coli enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 44
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15723541
- Full Text :
- https://doi.org/10.1021/bi048657a