Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid.

Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.     

Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.     

The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.     

A simplified procedure for the purification of rat liver tyrosine aminotransferase.

Rat liver tyrosine aminotransferase was purified by chromatography on CM-Sephadex C-50 and DEAE-cellulose, (NH4)2SO4 fractionation and gel filtration on Sephadex G-200. Livers from 400 rats can be easily worked up by this procedure. Furthermore, this purification method has the advantage that hepatic tryptophan 2,3-dioxygenase, which, like tyrosine aminotransferase, is induced by glucocorticosteroids, can be purified from the same homogenate. Tyrosine aminotransferase purified by this method was shown to be specific for 2-oxoglutarate. Its subunits have a molecular weight of 45 000. The following "apparent" Michaelis constants were determined: L-tyrosine, 1.7 X 10(-3) M; 2-oxoglutarate, 5.9 X 10(-4) M; and pyridoxal 5'-phosphate, 2.1 X 10(-6) M. Tyrosine aminotransferase, depleted of its cofactors, binds 4 molecules of pyridoxal 5'-phosphate per 90 000 daltons with a KA of 2.2 X 10(5) M-1.     

Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase

The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.     

Spectrophotometric determination of pyridoxal and pyridoxal 5′-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay

A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.     

Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids

Thermodynamic and kinetic parameters for Schiff base formation of pyridoxal 5'-phosphate and pyridoxal with epsilon-aminocaproic acid as well as of pyridoxal 5'-phosphate with L-serine were obtained in 0.1 M sodium pyrophosphate buffer as a function of temperature. Changes in enthalpy, which were determined by direct microcalorimetry, were small at 25 degrees C, but varied strongly with pH for the reaction of pyridoxal 5'-phosphate with the amino acids. In contrast to the fast Schiff base formation of pyridoxal 5'-phosphate, a very slow reaction was found for pyridoxal and epsilon-aminocaproic acid concomitant with a larger change in enthalpy. By preventing hemiacetal formation the phosphate moiety plays a crucial role.     

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.     

Purification and characterization of yeast L-kynurenine aminotransferase with broad substrate specificity

L-Kynurenine aminotransferase [L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7] has been purified to homogeneity and crystallized from cell-free extracts of a yeast, Hansenula schneggii, grown in a medium containing L-tryptophan as an inducer. The enzyme has a molecular weight of about 100,000 and consists of two subunits identical in molecular weight (52,000). The enzyme exhibits absorption maxima at 280, 335, and 430 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme-bound pyridoxal 5'-phosphate shows negative circular dichroic extrema, in contrast with other pyridoxal 5'-phosphate acting on L-amino acids. In addition to L-kynurenine and alpha-ketoglutarate, which are the most preferred substrates, a large number of L-amino acids and alpha-keto acids can serve as substrates; the extremely broad substrate specificity is the most characteristic feature of this yeast enzyme. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. Certain dicarboxylic acids such as adipate and pimelate act as competitive inhibitors. Addition of various substrate amino acids to the culture medium results in the inductive formation of aminotransferases which are immunochemically indistinguishable from L-kynurenine aminotransferase.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Pyridoxine-derived B6 vitamers and pyridoxal 5'-phosphate-binding proteins in cytosolic and nuclear fractions of HTC cells

The nuclear fraction of rat hepatoma-derived HTC cells contained approximately 8% of the total cellular pyridoxal 5'-phosphate. HTC cells were able to metabolize [3H]pyridoxine to coenzymatically active pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. As HTC cells did not have any demonstrable pyridoxine-5'-phosphate oxidase activity, the conversion of pyridoxine to pyridoxal 5'-phosphate must have taken place by a nonconventional route. The ratio of pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate in the nonnuclear fraction of HTC cells was approximately 1:1, whereas in the nuclear fraction it was approximately 17:1, indicating that there was selective acquisition of pyridoxal 5'-phosphate by the nucleus. With the aid of a monoclonal antibody specific for the 5'-phosphopyridoxyl group, it was shown that there was one major pyridoxal 5'-phosphate-binding protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved nucleoplasmic extract of HTC cells. This finding was confirmed by radioautography of an SDS-PAGE-resolved nucleoplasmic extract obtained from cells grown in a medium containing [3H]pyridoxine. Isoelectric focusing followed by SDS-PAGE also indicated the presence of one major pyridoxal 5'-phosphate-binding protein in the nucleoplasmic extract of HTC cells having a relatively high isoelectric point (approximately 7). Data were obtained indicating that the protein might exist in a higher molecular weight form, probably a dimer. Currently, these findings constitute virtually all of the available information on vitamin B6 and the cell nucleus.     

Solubilization of 1,25-dihydroxyvitamin D3 receptor with pyridoxal 5'-phosphate from hen intestinal mucosa

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5'-phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH)2D3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (greater than 90%) of the mucosal 1,25-(OH)2D3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5'-phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH)2D3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH)2D3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.     

Characterization of pyridoxine auxotrophs of Escherichia coli: serine and pdxF mutants

At least six phenotypically distinct classes of mutants of Escherichia coli which require serine or pyridoxine or both can be isolated. Three of the six classes lack 3-phosphoserine-2-oxoglutarate aminotransferase. One of these classes contains WG5, a mutant previously characterized as containing the pdxF5 allele. The aminotransferase isolated from this mutant has been compared to that isolated from wild-type E. coli and found to have apparently normal affinity for pyridoxal 5'-phosphate, but reduced affinity for pyridoxamine 5'-phosphate.     

Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid

Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.     

D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).