PMID:7556224
| Citation |
Graber, R, Kasper, P, Malashkevich, VN, Sandmeier, E, Berger, P, Gehring, H, Jansonius, JN and Christen, P (1995) Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme. Eur. J. Biochem. 232:686-90 |
|---|---|
| Abstract |
The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity. |
| Links | |
| Keywords |
Aspartate Aminotransferases/chemistry; Aspartate Aminotransferases/genetics; Aspartate Aminotransferases/metabolism; Binding Sites; Carboxy-Lyases/chemistry; Carboxy-Lyases/genetics; Carboxy-Lyases/metabolism; Crystallography, X-Ray; Electrochemistry; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen Bonding; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Pyridoxal Phosphate/chemistry; Pyridoxal Phosphate/metabolism; Substrate Specificity; Thermodynamics |
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