Abstract
Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.
Highlights
The pyridoxal 5Ј-phosphate (PLP)1-dependent enzymes that catalyze transformations of amino acids constitute a few families of evolutionarily related enzymes [2]
The cofactor shuttles between the PLP and the pyridoxamine 5Јphosphate (PMP) forms
Steady-state aspartate aminotransferase activity was measured in a coupled assay with malate dehydrogenase in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C in the presence of 20 mM 2-oxoglutarate plus 20 mM or 150 mM L-aspartate for wild-type enzyme or mutant Aspartate aminotransferase (AspAT), respectively
Summary
Site-directed Mutagenesis and Purification of Wild-type and Mutant AspATs—Oligonucleotide-directed mutagenesis of the wild-type aspC gene of Escherichia coli inserted into the BS M13 vector [17] was performed with the mutagenesis kit of Bio-Rad [18] and the oligonucleotides GC CAC ATT TAC TTT ACC AGA AGC and GA GTA GTT AGC TTT AAT CGC CGC T for the R386K and the R292K mutation, respectively. The calculated values of kcat/Km were in good agreement with the values measured at low substrate concentrations ([S0] ϽϽ Km), where kobs/[S0] gives directly kcat/Km. Steady-state aspartate aminotransferase activity was measured in a coupled assay with malate dehydrogenase in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C in the presence of 20 mM 2-oxoglutarate plus 20 mM or 150 mM L-aspartate for wild-type enzyme or mutant AspATs, respectively. Determination of Dissociation Equilibrium Constants for Competitive Inhibitors and of pKЈa of Internal Aldimine by Spectral Titration— Spectral titrations of the PLP form of the wild-type and mutant enzymes were performed at 25 °C in 20 mM sodium phosphate. Assay for Newly Generated Activities toward Amino Acids—Mutant AspATs and wild-type enzyme (0.9 mM subunit concentration) were incubated in 50 mM 4-methylmorpholine, pH 7.5, at 25 °C with different
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