Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.     

Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectral tuning of avian violet- and ultraviolet-sensitive visual pigments

The violet- and ultraviolet-sensitive visual pigments of birds belong to the same class of pigments as the violet-sensitive (so-called blue) pigments of mammals. However, unlike the pigments from mammals and other vertebrate taxa which, depending on species, have lambda(max) values of either around 430 nm or around 370 nm, avian pigments are found with lambda(max) values spread across this range. In this paper, we present the sequences of two pigments isolated from Humbolt penguin and pigeon with intermediate lambda(max) values of 403 and 409 nm, respectively. By comparing the amino acid sequences of these pigments with the true UV pigments of budgerigar and canary and with chicken violet with a lambda(max) value of 420 nm, we have been able to identify five amino acid sites that show a pattern of substitution between species that is consistent with differences in lambda(max). Each of these substitutions has been introduced into budgerigar cDNA and expressed in vitro in COS-7 cells. Only three resulted in spectral shifts in the regenerated pigment; two had relatively small effects and may account for the spectral shifts between penguin, pigeon, and chicken whereas one, the replacement of Ser by Cys at site 90 in the UV pigments, produced a 35 nm shortwave shift that could account for the spectral shift from 403 nm in penguin to around 370 nm in budgerigar and canary.     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the 'Genji' firefly, Luciola cruciata, was mutated with hydroxylamine to isolate mutant luciferases. Some of the isolated mutant enzymes produced different colors of light, ranging from green to red. Five such mutants, producing green (lambda max = 558 nm), yellow-orange (lambda max = 595 nm), orange (lambda max = 607 nm) and red light (lambda max = 609 and 612 nm), were analyzed. The mutations were found to be single amino acid changes, from Val239 to Ile, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser and His433 to Tyr respectively.     

Mycosporine-like amino acids (MAAs) profile of a rice-field cyanobacterium Anabaena doliolum as influenced by PAR and UVR

The mycosporine-like amino acid (MAA) profile of a rice-field cyanobacterium, Anabaena doliolum, was studied under PAR and PAR + UVR conditions. The high-performance liquid chromatographic analysis of water-soluble compounds reveals the biosynthesis of three MAAs, mycosporine-glycine (lambda (max) = 310 nm), porphyra-334 (lambda (max) = 334 nm) and shinorine (lambda (max) = 334 nm), with retention times of 4.1, 3.5 and 2.3 min, respectively. This is the first report for the occurrence of mycosporine-glycine and porphyra-334 in addition to shinorine in Anabaena strains studied so far. The results indicate that mycosporine-glycine (monosubstituted) acts as a precursor for the biosynthesis of the bisubstituted MAAs shinorine and porphyra-334. Mycosporine-glycine was under constitutive control while porphyra-334 and shinorine were induced by UV-B radiation, indicating the involvement of UV-regulated enzymes in the biotransformation of MAAs. It seems that A. doliolum is able to protect its cell machinery from UVR by synthesizing a complex set of MAAs and thus is able to survive successfully during the summer in its natural brightly lit habitats.     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.     

Experimental study of the excited-state properties and photostability of the mycosporine-like amino acid palythine in aqueous solution.

Characterization of the excited states of the mycosporine-like amino acid palythine (lambda(max) = 320 nm) in aqueous solutions was achieved experimentally. The low value for the photodegradation quantum yield, (1.2 +/- 0.2) x 10(-5), confirms that palythine is highly photostable in air saturated-aqueous solutions. Laser flash photolysis of acetone in the presence of palythine allowed for the observation of a transient spectrum which is consistent with the triplet-triplet absorption of palythine. Kinetic treatment of the transient signals yields a lifetime of the triplet state of ca. 9 micros and a triplet energy around 330 kJ mol(-1). The photoacoustic calorimetry results are consistent with non-radiative decay as the major fate of excited palythine. A comparison of the photodegradation quantum yields and photophysical properties of palythine with those previously determined for the other mycosporine-like amino acids, shinorine and porphyra-334, suggests that geometrical isomerization around the C=N bond may contribute to the rapid deactivation of this group of molecules.     

Enantiospecific change in products for aldose reductase-mediated reaction of glyceraldehyde with bound NADP+

Aldose reductase-mediated reaction of glyceraldehyde with enzyme-bound NADP+ gives different products depending on the enantiomer used. D-Glyceraldehyde reacts to form a chromophore (336 nm) similar to the covalent NADP-glycolaldehyde adduct characterized previously [Grimshaw et al. (1990) Biochemistry 29, 9936-9946]. L-Glyceraldehyde, however, reacts in a slow steady-state process to form an additional chromophore whose spectral properties (lambda max 290 nm, epsilon approximately 16,700 M-1cm-1) suggest that hydration of the nicotinamide 5,6-double bond has occurred. Several mechanisms are proposed to explain this unique stereoisomer-dependent change in reaction pathway.     

Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate-pyrocatechol

Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with lambda(max) of 580nm or coupling with 3-aminophenol to produce an orange yellow-colored product with lambda(max) of 460nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a lambda(max) of 490nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods.     

The "five-sites" rule and the evolution of red and green color vision in mammals

Amino acid changes S180A (S-->A at site 180), H197Y, Y277F, T285A, and A308S are known to shift the maximum wavelength of absorption (lambda max) of red and green visual pigments toward blue, essentially in an additive fashion. To test the generality of this "five-sites" rule, we have determined the partial amino acid sequences of red and green pigments from five mammalian orders (Artiodactyla, Carnivora, Lagomorpha, Perissodactyla, and Rodentia). The result suggests that cat (Felis catus), dog (Canis familiaris), and goat (Capra hircus) pigments all with AHYTA at the five critical sites have lambda max values of approximately 530 nm, whereas rat (Rattus norvegicus) pigment with AYYTS has a lambda max value of approximately 510 nm, which is accurately predicted by the five-sites rule. However, the observed lambda max values of the orthologous pigments of European rabbit (Oryctolagus cuniculus), white-tailed deer (Odocoileus virginianus), gray squirrel (Sciurus carolinensis), and guinea pig (Cavia procellus) are consistently more than 10 nm higher than the predicted values, suggesting the existence of additional molecular mechanisms for red and green color vision. The inferred amino acid sequences of ancestral organisms suggest that the extant mammalian red and green pigments appear to have evolved from a single ancestral green-red hybrid pigment by directed amino acid substitutions.      

Copper complexes at N- and C-site of ovotransferrin: quantitative determination and visible absorption spectrum of each complex

Copper complexes at the two sites of ovotransferrin (TF) differed markedly in the rate of Cu release by EDTA. During the reaction, lambda max of the remaining Cu-Tf complex shifted to red side, while the difference spectrum of FenCu2-nTf vs. FenTf in which the N-site had been preferentially occupied with Fe had lambda max at blue side from that of Cu2Tf, 440 nm. From these results, the intrinsic spectrum for Cu-complex at each site was assigned: lambda max 450 nm for N- and 430 nm for C-site. The differences in the release rate and the spectrum can be used for the identification of the two domains of Tf and for the analysis of metal-binding behavior of each site.     

Iron-bleomycin interactions with oxygen and oxygen analogues. Effects on spectra and drug activity.

Despite extensive structural dissimilarities, iron . bleomycin complexes and heme-containing oxygenases display remarkable similarities in binding oxygen antagonists and in spectral properties deriving from bound iron. Fe(II)-bleomycin reversibly forms a complex with either CO or isocyanide (lambda max = 384 and 497 nm, respectively), either of which interfere with its oxygen-dependent cleavage of DNA. A similar but paramagnetic complex forms with NO (lambda max = 470 nm; AN = 24 G). In contrast, cyanide enhances bleomycin activity against DNA. Complexes of bleomycin and FE(III), formed either by direct association or by autoxidation of the Fe(II) . bleomycin complex, exhibit indistinguishable EPR and visible spectra, which change characteristically with pH. At neutral pH, Fe(III) . bleomycin is a low spin complex (g = 2.45, 2.18, 1.89; lambda max = 365, 384 nm) and, at low pH, it is a high spin rhombic complex (geff = 9.4, 4.3; lambda max = 430 nm). These complexes are interconvertible (pK 4.3). Fe(II) . bleomycin oxidation, although reversible by spectral criteria, is accompanied by drug inactivation unless DNA is present.     

Color vision of the coelacanth (Latimeria chalumnae) and adaptive evolution of rhodopsin (RH1) and rhodopsin-like (RH2) pigments

The coelacanth, a "living fossil," lives at a depth of about 200 m near the coast of the Comoros archipelago in the Indian Ocean and receives only a narrow range of light at about 480 nm. To see the entire range of "color" the Comoran coelacanth appears to use only rod-specific RH1 and cone-specific RH2 visual pigments, with the optimum light sensitivities (lambda max) at 478 nm and 485 nm, respectively. These blue-shifted lambda max values of RH1 and RH2 pigments are fully explained by independent double amino acid replacements E122Q/A292S and E122Q/M207L, respectively. More generally, currently available mutagenesis experiments identify only 10 amino acid changes that shift the lambda max values of visual pigments more than 5 nm. Among these, D83N, E1220, M207L, and A292S are associated strongly with the adaptive blue shifts in the lambda max values of RH1 and RH2 pigments in vertebrates.     

Spectroscopic and kinetic characterization of nonenzymic and aldose reductase mediated covalent NADP-glycolaldehyde adduct formation

Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold.     

Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.

The catalytic-site thiol groups of papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were each labelled with the nitrobenzofurazan (Nbf) chromophore by reaction with 4-chloro-7-nitrobenzofurazan at pH 4.4. The electronic-absorption spectra of both labelled enzymes were determined in aqueous solution, in the pH ranges approx. 2-5 for S-Nbf-papain and approx. 3.3-8 for S-Nbf-actinidin, and for the latter also in 6 M-guanidinium chloride. The spectrum of S-Nbf-papain is characterized by lambda max. = 402 nm at pH 5 and by lambda max. = 422 nm at pH 2.18. The pH-dependent shift in lambda max. accompanies a pH-dependent change in A 430, the nature of which is consistent with its dependence on a single ionizing group with pKa 3.7. The spectrum of S-Nbf-actinidin is pH-independent in the pH range approx. 3.3-8 and is characterized by lambda max. = 413 nm. This absorption maximum shifts to 425 nm in 6M-guanidinium chloride. These results are discussed and related to those reported previously from studies on papain and actinidin with various reactivity probes. Despite the close similarity in the catalytic sites of papain and actinidin deduced from X-ray-diffraction studies, the considerable differences in their reactivity characteristics are mirrored by differences in their electric fields detected by the Nbf spectroscopic label. The microenvironment in the catalytic site of actinidin appears to favour the existence of ions significantly more than in the corresponding region in papain.     

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.     

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.     

Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium

Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography.