OF SITE-DIRECTED MUTAGENESIS AND ALTERNATIVE SUBSTRATES TO ASSIGN THE PROTOTROPIC GROUPS IMPORTANT TO CATALYSIS BY ESCHERICHIA COLI ASPARTATE AMINOTRANSFERASE

摘要

The pH dependence of Escherichia coli aspartate aminotransferase (AATase) has been investigated by the use of site-directed mutants and alternative substrates. Inhibition of the enzyme by CHES and variations in ionic strength are proposed to explain some of the qualitative differences in the published pH dependence of pig cytosolic AATase kinetics [Velick, S. F., and Vavra, J. (1962) J. Biol. Chem. 237, 2109-2122; Kiick, D. M., and Cook, P. F. (1983) Biochemistry 22, 375-382]. The pK(a) values of the basic limbs in the k(cat)/K-M profiles for the amino acids, L-Asp and L-cysteinesulfinate (L-CS), are identical, within error, to those of free substrates, (L-Asp, pK(a) = 9.6; L-CS, pK(a) 9.0). This pK(a) therefore is assigned to the alpha-amino group of the substrate. Replacement of the active site base, Lys-258, with the weaker base, gamma-thia-Lys, does not alter the intrinsic pK(a) for the profiles of the K-i values for the maleate-E . PMP complexes or the k(cat)/K-M(alpha-KG) values. The mutation Y225F results in an alkaline shift of the pK(a) in the k(cat)/K-M(alpha-KG) profile. This pK(a) is assigned to the C4' amino group of PMP. E. coli AATase, unlike pig cytosolic AATase, shows a pH dependence on k(cat) between pH 5 and 10 that arises from a change in the rate-determining step at pH extremes. C-alpha proton abstraction is partially rate-determining at neutral pH values, but not at pH extremes. The pH dependence of the H-2-C-alpha kinetic isotope effects demonstrates that ketimine hydrolysis, oxalacetate dissociation, or the alpha-KG half-reaction is rate-determining at the pH extremes.