Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme.
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.
Graber
R
R
Biochemisches Institut, Universität Zürich, Switzerland.
Kasper
P
P
Malashkevich
V N
VN
Sandmeier
E
E
Berger
P
P
Gehring
H
H
Jansonius
J N
JN
Christen
P
P
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D002262:Carboxy-Lyases; C019747:aspartate 4-decarboxylase