PubMed · 4408188
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S Vaisrub. 1974-09-16. Editorial: A different drummer.. https://doi.org/10.1001/jama.229.12.1639
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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 (gamma-thiahomolysine and gamma-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 approximately 3 kcal/mol relative to Quint; the 258-side-chain carboxylate variants stabilize E.PMP vs E.PLP by 5.8 kcal/mol relative to WT. The C alpha-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 C alpha proton abstraction for both sets of enzymes. The values of kcat/KM calculated for the 258-side-chain carboxylate variants from the Brønsted plot of Toney and Kirsch [(1989) Science 243, 1485-1488] are in close agreement with those observed. This "tethered" Brønsted analysis shows that the earlier reported poor reactivity of carboxylates in chemical rescue is due to electrostatic exclusion from the active site.
The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity.
The pyridoxal phosphate form of aspartate aminotransferase from Escherichia coli catalyzes the irreversible conversion of L-cysteine sulfinate to the pyridoxamine phosphate form of the enzyme, bisulfite, and pyruvate. The addition of L-cysteine sulfinate to a solution containing a high concentration of enzyme (approximately 10 microM) yields a rapidly appearing red color (lambda max = 520 nm) which decays with a rate constant which is only about 1% of kcat (2-3 s-1 versus 250 s-1 at 15 degrees C, pH 7). The red color can be assigned to the quinonoid form of the enzyme substrate complex, which accumulates under these single turnover conditions. The rate of decay of this species is dependent on that for the decomposition of beta-sulfinylpyruvate (beta-SP), the initial product of the reaction between aspartate aminotransferase and L-cysteine sulfinate. Trapping beta-SP with morpholine or malate dehydrogenase plus NADH abolishes the transient red color; therefore, the intermediate accumulates by virtue of the reverse reaction of beta-SP with the pyridoxamine phosphate form of the enzyme. The association and dissociation rate constants of beta-SP with the pyridoxamine-5'-phosphate form of the enzyme are 2 x 10(7) M-1 s-1 and 400 s-1, respectively, at 15 degrees C. No red transient species is observed under these conditions when aspartate is substituted for L-cysteine sulfinate.