Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase

Y Inoue, S Kuramitsu, K Inoue, H Kagamiyama, K Hiromi, S Tanase, Y Morino
1989 Journal of Biological Chemistry  
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
more » ... 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.
pmid:2498335 fatcat:p4pz4iqtlffxnmowlkco3kwtk4