Examining the Structural and Chemical Flexibility of the Active Site Base, Lys-258, of Escherichia coli Aspartate Aminotransferase by Replacement with Unnatural Amino Acids

Abstract

The active site base, Lys-258, of Escherichia coli aspartate aminotransferase (AATase) was mutated to a unique cysteine for chemical modification in a cysteine-free AATase [Quint; Gloss et al. (1992) Biochemistry 31, 32-39]. Two homolysine analogues (γ-thiahomolysine and y-dithiohomolysine) and the carboxylate series [glutamate, (carboxymethyl)cysteine, and (carboxyethyl)cysteine] were prepared. The kcat and kcat/Km values for amino acids exhibited by the 258-homo-Lys AATases are ca. 1000-fold less than those of Quint. However, the kcat/KM values for keto acids are only reduced by 2.5-10-fold. The 258-side-chain carboxylates bind amino acids 1-4 orders of magnitude more tightly than Quint, but have first-order catalytic rate constants 5-7 orders of magnitude less than Quint. The 258-homo-Lys AATases stabilize the E*PLP vs the E*PMP species by ~3 kcal/mol relative to Quint; the 258-side-chain carboxylate variants stabilize vs E*PLP by 5.8 kcal/mol relative to WT. The Ca-2H kinetic isotope effects for the reactions of 258-homo-Lys and 258-side-chain carboxylate AATases demonstrate that transaldimination is equally or more rate-determining than Ca proton abstraction for both sets of enzymes. The values of kcat/Km calculated for the 258-side-chain carboxylate variants from the Brpnsted plot of Toney and Kirsch [(1989) Science 243, 1485-1488] are in close agreement with those observed. This “tethered” Brpnsted analysis shows that the earlier reported poor reactivity of carboxylates in chemical rescue is due to electrostatic exclusion from the active site. © 1995, American Chemical Society. All rights reserved.