Functional role of the amino-terminal mobile segment in catalysis by porcine cytosolic aspartate aminotransferase. Critical importance of Val17 and Phe18 for productive binding of substrates.
A notable feature of porcine cytosolic aspartate aminotransferase is the closure of the active site cleft by a mobile amino-terminal segment (residues 15-40) upon binding substrate. The functional roles of Val17 and Phe18, residues that are part of the mobile loop, have been studied in the site-directed mutants in which the size and hydrophobic nature of these residues have been changed. Absorption, circular dichroism spectra, susceptibility to protease 401, and thermal stability did not differ appreciably between wild type and mutant enzymes. In the overall transamination between aspartate and 2-oxoglutarate, V17A represented a typical Km mutant while V17I retained the substrate binding affinity fairly well. In contrast, replacement of Phe18 by Ala resulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y, and F18H showed a moderate decrease in kcat and a considerable increase in Km values. Single-turnover reactions with four individual substrates yielded analogous results to those obtained for the overall reaction and, in addition, revealed that k/Kd values of mutants F18A and F18H were over 10 times lower for C5 substrates (glutamate and 2-oxoglutarate) than those for C4 substrates (aspartate and oxalacetate). All mutant enzymes showed variously increased Kd values for substrate analogs such as 2-methylaspartate, succinate, and glutarate. 1H NMR observations of F18H, in which His18 served as a built-in probe, were in accord with the behavior that would be expected from the conformational transition. We conclude that, although Val17 and Phe18 may not be essential for catalysis, the presence of a bulky residue of appropriate size at each position is critical for productive binding of substrate.
Nishimura
K
K
Department of Biochemistry, Kumamoto University School of Medicine, Japan.
Higaki
T
T
Okamura
H
H
Tanase
S
S
D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D013329:Structure-Activity Relationship; D013552:Swine; D014633:Valine
D010649:Phenylalanine; D001219:Aspartate Aminotransferases; D014633:Valine