Role of an active site residue analyzed by combination of mutagenesis and coenzyme analog.
Asp222 of aspartate aminotransferase is an active-site residue which interacts with the pyridine nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP). The roles of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase have previously been explored by site-directed mutagenesis. These studies confirmed that a negatively charged residue at position 222 is essential for catalysis, but the reason for this remained speculative. In the present studies, the roles of Asp222 were clarified experimentally by analyzing the mutant D222A enzyme (Asp222 replaced by Ala) reconstituted with the coenzyme analog N(1)-methylated PLP (N-MePLP). Spectroscopic and kinetic analyses showed that Asp222 stabilizes the protonated N(1) of PLP, raising the pKa value of N(1) by more than five units, in the active site of AspAT. The positive charge at N(1) accelerates abstraction of the alpha-proton from the amino acid substrate, stabilizing the transition state by 1.4 to 4.5 kcal.mol-1 in the reaction with aspartate. X-ray crystallographic (2.0 A resolution) and CD spectroscopic studies suggest that the coenzyme analog is not held in a proper orientation within the active site of D222A (N-MePLP). This may account for the finding that the catalytic activity was recovered only partially by the reconstitution of D222A with N-MePLP. These results fully support the following postulated role of Asp222: the negative charge of Asp222 stabilizes the positive charge at N(1) of PLP and thereby enhances the function of PLP as an electron sink.
Yano
T
T
Department of Medical Chemistry, Osaka Medical College, Japan.
Hinoue
Y
Y
Chen
V J
VJ
Metzler
D E
DE
Miyahara
I
I
Hirotsu
K
K
Kagamiyama
H
H
D001219:Aspartate Aminotransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000737:chemistry; D011994:Recombinant Proteins; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases