Decreasing the Basicity of the Active Site Base, Lys-258, of Escherichia Coli Aspartate Aminotransferase by Replacement with γ-Thialysine

Abstract

Alkylation of the K258C mutant of the wild-type aspartate aminotransferase (AATase) with bromoethylamine to give γ-thialysine 258 was complicated by partial reaction with the five native cysteines [Planas, A., & Kirsch, J. F. (1991) Biochemistry 30, 8268-8276]. This problem is now overcome by carrying out the alkylation with K258CQ, 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, K258CQ-EA, have been examined and compared to those of WT and Quint. The replacement of Lys-258 by γ-thia- Lys results in an acidic shift of 1.3 pH units in the pKa of the internal aldimine. The Ca hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(kCat/KASPMDkCat, respectively. Replacement of Lys-258 by the weaker base, γ-thia-Lys, increases these values to 3.3 and 2.6, respectively The changes of K258CQ-EA in ligand affinities and the keto acid half-reaction are minor; however, the kCat/KM values for amino acids are decreased by an order of magnitude. The Kb values for PMP of K258CQ-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 K258CQ-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 K258CQ-EA is 1.4 kcal/mol more stable than that of Quint, while the corresponding E-PMP complexes are equally stable. © 1995, American Chemical Society. All rights reserved.