Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase.
Substitution of a lysyl residue for Arg-386 of Escherichia coli aspartate aminotransferase resulted in an extensive decrease in Vmax values (0.8% with the aspartate-2-oxoglutarate pair and 0.2% with the glutamate-oxalacetate pair, compared with the corresponding values for the wild-type enzyme). Kinetic analysis of the four sets of half-reactions, the pyridoxal form of the enzyme with aspartate or glutamate and the pyridoxamine form with 2-oxoglutarate or oxalacetate, allowed us to define the independent effect of the mutation on the reactivity of each substrate. Decrease in the first order rate constant (kmax) was more pronounced in the reactions with five-carbon substrates (glutamate and 2-oxoglutarate) than in those with four-carbon substrates (aspartate and oxalacetate), while the increase in the apparent dissociation constant (Kd) was greater for four-carbon substrates than for five-carbon substrates. The decrease of overall catalytic efficiency as judged by the values, kmax/Kd, was more pronounced in the reactions with five-carbon substrates than in those with four-carbon substrates. Affinities for substrate analogs such as succinate, glutarate, 2-methylaspartate, and erythro-3-hydroxyaspartate, were also considerably decreased by the mutation of the enzyme. These findings indicate that the side chain of the lysyl residue, although it bears a positive charge similar to that of the arginyl residue, is not structurally adequate for the productive binding of a substrate during catalysis.
Inoue
Y
Y
Department of Medical Chemistry, Osaka Medical College, Japan.
Kuramitsu
S
S
Inoue
K
K
Kagamiyama
H
H
Hiromi
K
K
Tanase
S
S
Morino
Y
Y
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001219:Aspartate Aminotransferases / Q000235:genetics*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D009154:Mutation; D016202:N-Methylaspartate; D013379:Substrate Specificity
C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D016202:N-Methylaspartate; D001120:Arginine; D001219:Aspartate Aminotransferases; D008239:Lysine