Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase (original) (raw)
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Biochemistry, 1989
Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17 000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphonoacetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [SI,,. Gln-137, 'This work was supported by Grants DK1429 and GM26237 from the *To whom correspondence should be addressed. National Institutes of Health. (1973), Schachman (1974), Kantrowitz et al. (1980a,b), and Kantrowitz and Lipscomb (1988)], exhibits positive cooperativity for both substrates (Gerhart & Pardee, 1962; Bethel1 et a]., 1968), and its activity is inhibited by CTP and activated by ATP, the end products of the pyrimidine and purine pathways, respectively. The enzyme is composed of three regulatory dimers (Le., regulatory subunits) and two catalytic trimers (Le., catalytic subunits). The regulatory subunit binds CTP and ATP but is devoid of catalytic activity while the isolated catalytic subunit exhibits no homotropic cooperativity
Journal of Biological Chemistry, 1997
Arg 386 and Arg 292 of aspartate aminotransferase bind the ␣ and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The k cat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25°C were 0.5, 2.0, and 0.03 s ؊1 for the R292K, R386K, and R292K/ R386K mutations, respectively, k cat 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 Arg 292 and Arg 386 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 -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 (k cat ؍ 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 3 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.
Biochemistry, 2006
An X-ray diffraction study to 2.0 Å resolution shows that this enzyme, ATCase, is in the T-state (the c 3 to c 3 distance is 45.2 Å) when ATCase is bound to carbamyl phosphate (CP) and to L-alanosine (an analogue of aspartate). This result strongly supports the kinetic results that alanosine did not inhibit the carbamylation of aspartate in the normal reaction of native ATCase plus CP and aspartate Biochemistry 24, 7182-7187]. The structure further reveals that the phosphate of CP is 4 Å away from its known position in the R-state and is in the T-state position of P i in a recent study of ATCase complexed with products, phosphate (P i ) and N-carbamyl-L-aspartate [Huang, J., and Lipscomb, W. N. (2004) Biochemistry 43, 6422-6426]. Moreover, the alanosine position in this T-state is somewhat displaced from that expected for its analogue, aspartate, from the R-state position. The relations of these structural aspects to the kinetics are presented.
Biochemistry, 2011
P yridoxal 5 0-phosphate (PLP) enzymes are unequaled in the variety of reaction types they catalyze, allowed by the cofactor's ability to resonance stabilize carbanionic intermediates (Figure 1A). 1À3 Indeed, considering the nearly universal invocation of the quinonoid resonance form of carbanionic intermediates in PLP enzyme mechanisms, one would expect that all enzymes allow the full potential of the electron sink by protonation of the pyridine ring. However, this is not the case. The X-ray structures of several PLP enzymes implicate different pyridine ring protonation states. 4À6 At one extreme, aspartate aminotransferase (AAT), a representative fold type I enzyme, has an aspartic acid residue (Asp222; solution pK a ∼ 4) that fully protonates the pyridine nitrogen (solution pK a ∼ 6.3 as a Schiff base 7) of PLP 8 to form a salt bridge 9 (Figure 1B). In the middle is alanine racemase (ALR), a fold type III enzyme, which has a positively charged arginine residue (Arg219; solution pK a ∼ 13) that forms a hydrogen bond to the pyridine N. 5 At the other extreme are enzymes like O-acetylserine sulfhydrylase (OASS), a fold type II enzyme with a serine residue (Ser272; solution pK a ∼ 15) that forms a hydrogen bond to the pyridine N. 6 This interaction between the pyridine ring and a serine residue is likely to leave the pyridine nitrogen unprotonated (Figure 1B). Recent experimental and computational studies indicate that the protonated imino group of the external aldimine intermediate, through electrostatic and resonance stabilization of the carbanion (Figure 1A), plays a primary role in catalysis. 1,10À13 We synthesized an isosteric carbocyclic analogue of PLP, 1-deazapyridoxal 5 0-phosphate (deazaPLP), to determine the extent to which various PLP enzymes require the electron sink properties of the pyridine ring. 14 DeazaPLP, lacking both the potential for protonation and the greater electronegativity of nitrogen versus carbon in the aromatic ring, is expected to be a very poor cofactor with fold type I enzymes, and less poor with fold type II and III PLP enzymes. This prediction is true in the case of aspartate aminotansferase. We report here the crystal structures of the internal aldimine and the stable L-aspartate external aldimine of Escherichia coli AAT reconstituted with deazaPLP.
Journal of Biological Chemistry, 1997
Arg 386 and Arg 292 of aspartate aminotransferase bind the ␣ and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The k cat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25°C were 0.5, 2.0, and 0.03 s ؊1 for the R292K, R386K, and R292K/ R386K mutations, respectively, k cat 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 Arg 292 and Arg 386 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 -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 (k cat ؍ 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 3 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.