DECREASING THE BASICITY OF THE ACTIVE-SITE BASE, LYS-258, OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE BY REPLACEMENT WITH GAMMA-THIALYSINE

Alkylation of the K258C mutant of the wild-type aspartate aminotransferase (AATase) with bromoethylamine to give gamma-thialysine 258 was complicated by partial reaction with the five native cysteines [Planas, A., and Kirsch, J. F. (1991) Biochemistry 30, 8268-8276]. This problem is now overcome by carrying out the alkylation with K258C(Q), in which Cys-258 is a unique cysteine residue in Quint, an engineered AATase in which the five cysteines have been converted to alanine [Gloss, L. M., et al. (1992) Biochemistry 31, 32-39]. The kinetics and spectral properties of the resulting enzyme, K258C(Q)-EA, have been examined and compared to those of WT and Quint. The replacement of Lys-258 by gamma-thia-Lys results in an acidic shift of 1.3 pH units in the pK(a) of the internal aldimine. The C-alpha hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(k(cat)/K-M(Asp)) and D-kcat, respectively. Replacement of Lys-258 by the weaker base, gamma-thia-Lys, increases these values to 3.3 and 2.6, respectively The changes of K258C(Q)-EA in ligand affinities and the keto acid half-reaction are minor; however, the k(cat)/K-M values for amino acids are decreased by an order of magnitude. The K-D values for PMP of K258C(Q)-EA and Quint are equal to each other (0.2 nM) and are 7-fold lower than that of WT. These combined effects are illustrated in the free energy diagrams of the reaction with L-Asp with K258C(Q)-EA, relative to WT (and Quint). The E . PLP and E . PMP complexes of Quint are 0.9 and 1.1 kcal/mol, respectively, more stable than those of WT. The E . PLP form of K258C(Q)-EA is 1.4 kcal/mol more stable than that of Quint, while the corresponding E . PMP complexes are equally stable.