The L-aspartate oxidase reported to be present in higher plants is actually glutamic oxaloacetic transaminase (original) (raw)
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European Journal of Biochemistry, 1996
This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant ( K J equal to 1.4 pM. The enzyme binds FAD by a simple second-order process with K, 0.67 pM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
L‐Aspartate Oxidase from Escherichia coli
European Journal of Biochemistry
This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant ( K J equal to 1.4 pM. The enzyme binds FAD by a simple second-order process with K, 0.67 pM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
The Reaction of beta-Chloroglutamic Acid with Glutamate-Aspartate Transaminase
European Journal of Biochemistry, 1968
Porcine glutamate-aspartate transaminase catalyzes a @-elimination reaction with both the threo-and erythro-isomers of 8-chloroglutamate ; chloride, ammonia, and a-ketoglutarate are formed in equimolar amounts. The latter product was characterized as the 2,4-dinitrophenylhydrazone and by catalytic hydrogenation of this derivative to glutamic acid.
The Isozymes of Glutamate-Aspartate Transaminase
Journal of Biological Chemistry, 1970
The binding of ar-keto acids and other dicarboxylic acids to the pyridoxal form of the isozymes of pig heart glutamateaspartate transaminase (EC 2.6.1.1) can be studied spectrophotometrically because the absorption of the resultant complexes differs from that of the free enzymes. Spectroscopic and catalytic rate data indicate a correlation of the inhibition of transamination with the affinity of the dicarboxylic acids for the pyridoxal form of the enzymes. Maleate and succinate are the most effective inhibitors of mitochondrial transaminase, whereas succinate, maleate, glutarate, and adipate inhibit the supernatant isozyme. The amino acid erythro-/3-hydroxy-r.-aspartate forms distinctive binary complexes of unique absorption maxima with either isozyme; the equilibria between these complexes are pH insensitive. Competition of dicarboxylic acids with erythro-/3-hydroxy-Laspartate shows that inhibition by 4-or 5-carbon dicarboxylic acids is accomplished through competition with the amino acid for the pyridoxal (aldimine) and pyridoxamine forms of the supernatant isozyme. In contrast, the mitochondrial isozyme is more highly affected by 4-carbon inhibitors. They compete with the amino acid for the pyridoxal form of the enzyme and also form ternary complexes of the nature enzyme-substrate-dicarboxylic acid. Combination of the keto acid with the enzyme shifts the proton ionization (pK) of the active site chromophore (bound pyridoxal phosphate) to higher pH values in both transaminases. Oxalacetate is much more effective than o(ketoglutarate in the mitochondrial isozyme. Differences for each isozyme in the effect of the pH on the aspartate-arketoglutarate and glutamate-oxalacetate transaminations are explained by preferential binding of oxalacetate to the mitochondrial enzyme and of either a-ketoglutarate or oxalacetate to the supernatant isozyme. Product and substrate inhibition have been used to study functional isozyme variations in an effort to discover the physio
Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue
Journal of Biological Chemistry, 1976
Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN'%O and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 & 0.08 and k = 146 * 5 M-k', which are similar to the values determined for carbamylation of the NH,terminal groups of human hemoglobin (
Journal of Enzyme Inhibition and Medicinal Chemistry, 2001
Aspartate aminotransferase (AAT, EC 2.6.1.1) catalyses the transamination of L-asparate to oxaloacetate. It has been reported that AAT from different plant sources can catalyse the transamination of other compounds structurally similar to the natural substrates. Specificity and kinetic studies were performed with two aspartate aminotransferase isoenzymes (AAT-1 and AAT-2) from leaves of Lupinus albus L. cv Estoril using different amino donors and acceptors. Both isoenzymes showed residual activity for some of the substrates tested. Competitive inhibition was found with most of the structural analogues which is typical of a ping-pong bi-bi kinetic mechanism. It was found that both isoenzymes can use 2-amino-4-methoxy-4-oxobutanoic acid as amino donor. AAT-2 uses 2-amino-4-methoxy-4-oxobutanoic acid at a similar rate as L-aspartate but AAT-1 uses this substrate at a slower rate. The use of this amino donor by AAT isoenzymes has not been reported previously, and our results indicate structural differences between both isoenzymes.
Comparative biochemistry and physiology. B, Comparative biochemistry
1. D-Amino acid oxidase (D-AAO) oxidizes: D-Met, D-Pro, D-Phe, D-Tyr, D-Ile, D-Leu, D-Ala and D-Val. D-Ser, D-Arg, D-His, D-norleucine and D-Trp are oxidized at a low rate. D-Ornithine, cis-4-hydroxy-D-proline, D-Thr, D-Trp-methyl ester, N-acetyl-D-Ala and D-Lys are oxidized at a very low rate. 2. D-Asp, D-Glu and their derivatives, Gly and all the L-amino acids are not oxidized (or are at a rate which is undetectable). 3. Among all D-amino acids, D-Met is the most highly oxidized compound. The Km value is 1.7 mM. 4. D-Aspartate oxidase (D-Aspo) either purified from Octopus vulgaris or from beef kidney oxidizes only D-Asp, D-Glu and their following derivatives: D-Asn, D-Gln, D-Asp-dimethyl-ester and N-methyl-D-Asp. 5. However, D-Pro, D-Leu, D-Ala and D-Met, are also oxidized by this enzyme, but at a very low rate (between 0.2 and 0.6% of D-Asp). 6. All other D-amino acids, glycine and all the L-amino acids are not oxidized. 7. Under experimental conditions, 1 U of D-AAO is able to t...
European Journal of Biochemistry, 1996
L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with K,, values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate :fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.
The Function of Ascorbate Oxidase in Tobacco
PLANT PHYSIOLOGY, 2003
The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, l-galactono-␥-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA ; fax 01582-763010.