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., & 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 gamma-thia-Lys results in an acidic shift of 1.3 pH units in the pKa of the internal aldimine. The C alpha hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(kcat/KMAsp) and Dkcat, 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 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 KD 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gloss
L M
LM
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Kirsch
J F
JF
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives*; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; C000204:S-2-aminoethyl cysteine; D003903:Deuterium; D001219:Aspartate Aminotransferases; D008239:Lysine; D003545:Cysteine