Aldimine Bond Research Articles

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5'-phosphate-dependent protein acting as a haemolytic enzyme.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris "Golden Beet") showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris "Altamo") plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation

The mechanism of pyridoxal phosphate (PLP) binding to apotryptophanase was investigated using stopped-flow kinetics with wild type (WT), W330F and C298S mutants. Based on the dependence of the rate constants on PLP concentrations for the fast and slow phases detected, two mechanistic schemes were proposed. For the WT and C298S mutant, the slow process is due to an isomerization of the aldimine complex after its formation, and not to the binding to an alternative conformation of the apoenzyme, which is the case proposed for the W330F mutant. It is suggested that during the cold inactivation process a conformational change precedes the aldimine bond cleavage. For the W330F apotryptophanase, another conformational change occurs subsequent to the aldimine bond cleavage.

Cold inactivation and dissociation into dimers of Escherichia coli tryptophanase and its W330F mutant form

The kinetics and mechanism of reversible cold inactivation of the tetrameric enzyme tryptophanase have been studied. Cold inactivation is shown to occur slowly in the presence of K + ions and much faster in their absence. The W330F mutant tryptophanase undergoes rapid cold inactivation even in the presence of K + ions. In all cases the inactivation is accompanied by a decrease of the coenzyme 420-nm CD and absorption peaks and a shift of the latter peak to shorter wavelengths. The spectral changes and the NaBH 4 test indicate that cooling of tryptophanase leads to breaking of the internal aldimine bond and release of the coenzyme. HPLC analysis showed that the ensuing apoenzyme dissociates into dimers. The dissociation depends on the nature and concentration of anions in the buffer solution. It readily occurs at low protein concentrations in the presence of salting-in anions Cl −, NO 3 − and I −, whereas salting-out anions, especially HPO 4 2−, hinder the dissociation. K + ions do not influence the dissociation of the apoenzyme, but partially protect holotryptophanase from cold inactivation. Thus, the two processes, cold inactivation of tryptophanase and dissociation of its apoform into dimers exhibit different dependencies on K + ions and anions.

Immobilization of DNA in Polyacrylamide Gel for the Manufacture of DNA and DNA–Oligonucleotide Microchips

Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino–DNA and amino–oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

Chemical methods of DNA and RNA fluorescent labeling.

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.

Crystal structures and solution studies of oxime adducts of mitochondrial aspartate aminotransferase.

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10(-8) M to 10(-6) M depending on the nature of the side-chain group. The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the omega-carboxylate of the inhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L-Asp and L-Glu are essential for stabilizing the closed form of aspartate aminotransferase.

Subunit Assembly in the Tryptophan Synthase α2β2 Complex: STABILIZATION BY PYRIDOXAL PHOSPHATE ALDIMINE INTERMEDIATES

This work is aimed at understanding subunit assembly in the tryptophan synthase alpha 2 beta 2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the beta 2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the beta 2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T alpha 2 beta 2 complex is inactive in reactions catalyzed by the beta 2 subunit but retains activity in the reaction catalyzed by the alpha subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T beta 2 subunit and alpha 2 beta 2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit dissociates more readily from the K87T beta 2 subunit than from the wild type beta 2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T beta 2 subunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T beta 2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type beta 2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type beta 2 subunit and by the external aldimine with L-serine in the K87T beta 2 subunit may further stabilize interaction with the alpha subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta 2 subunit.

A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase.

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.

The active site of Sulfolobus solfataricus aspartate aminotransferase

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase.

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5'-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.

Bioorganic chemistry of rhodopsins

Abstract

Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure

Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: l-aspartate + 2-oxoglutarate ⇋ oxaloacetate + l-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 Å resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies. The enzyme is composed of two identical subunits, which consist of two domains. The coenzyme is bound to the larger domain and is situated in a pocket near the subunit interface. The proximal and distal carboxylate group of dicarboxylic substrates are bound to Arg386 and Arg292, respectively, the latter residue belonging to the adjacent subunit. These interactions largely determine the substrate specificity of the enzyme. They not only position the substrate for efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 Å closer to the coenzyme. The replacement of the ε-amino group of Lys258 by the α-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by ~30 °. The released ε-amino group of Lys258 serves as a proton acceptor/donor in the 1,3-prototropic shift producing the ketimine intermediate. At this stage, or after hydrolysis of the ketimine bond, the coenzyme rotates back to an orientation between that in the “external” aldimine intermediate and that in the pyridoxal form. Throughout this process, the protonated pyridine nitrogen atom maintains a hydrogen bond to the β-carboxylate group of Asp222. Upon formation of the pyridoxamine form, the small domain moves back to its original position. The proposed mechanism is compatible with the known kinetic and stereochemical features of enzymic transamination.

10] Pyridoxal phosphate-binding site in enzymes: Reduction and comparison of sequences

Publisher Summary This chapter illustrates pyridoxal phosphate-binding site in enzymes. In pyridoxal phosphate-dependent enzymes, the formyl group at position 4 of pyridoxal-P forms an aldimine bond with the ɛ -amino group of a specific lysyl residue. Involvement of a single particular lysyl residue indicates that there is a specific domain structure favorable for stabilizing the aldimine bond which, otherwise, is readily hydrolyzed. X-Ray analyses of both cytosolic and mitochondrial isoenzymes of aspartate aminotransferase, representative pyridoxal-P-dependent enzymes, provide details of interaction between pyridoxal-P and the enzyme side chains in the coenzyme-accommodating active site. Only the Re side of the aldimine bond is open to solvent, the Si face being shielded from solvent. This stereospecific status of the bound coenzyme has been remarkably identical among a variety of pyridoxal-P-dependent enzymes. At neutral pH, pyridoxal-P absorbs at 390 nm, which shifts more or less to a longer wavelength (410-430 nm) upon the formation of an aldimine linkage with amino compounds or apoenzymes. Pyridoxal-P-aldimine bond is reduced by borohydride or borocyanohydride under mild conditions. The reduction renders the aldimine bond highly stable to hydrolysis. The resulting reduced enzyme is fragmented by either proteolytic or chemical cleavage, followed by isolation of a phosphopyridoxyl peptide and its structural determination.

A Monomeric Intermediate in the Reconstitution of Potato Apophosphorylase with Pyridoxal 5′-Phosphate1

The process of reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate (PLP) has been investigated to elucidate the structure-function relationship in phosphorylase [EC 2.4.1.1]. In time-course studies, the recovery of enzyme activity was found to be delayed, especially at low temperatures, compared with the binding of PLP to protein. On polyacrylamide gel electrophoresis, an intermediary enzyme species was detected which bound PLP in the same binding mode as the native holoenzyme does, but which had neither enzyme activity nor affinity for the substrate amylopectin. The intermediate is monomeric and is converted to the active holoenzyme with concomitant dimerization. NaBH4-treatment of the monomeric intermediate produced the reduced monomeric enzyme, which could be converted into the reduced dimeric enzyme with considerable enzyme activity. The findings support the view of the catalytic activity of phosphorylase requires the dimeric structure of protein. As in the animal enzyme, the aldimine bond between the PLP and the epsilon-amino group of the lysyl residue is also not essential for enzyme activity in plant phosphorylase.

The gecko visual pigment: a pH indicator with a salt effect.

1. Unlike rhodopsin, the extracted 521-pigment of the Tokay gecko (Gekko gekko) is pH-sensitive and changes its spectral absorbance in the pH range of 4.5-7.3. The colour change is reversible and pH can be employed to adjust the spectral maximum anywhere between 490 nm and its native location at 521 nm.2. The hypsochromic shift with increasing acidity is opposite to that expected for the protonation of the Schiff base nitrogen and suggests an action on the secondary system of interacting charges that have long been postulated to adjust vertebrate visual pigment colour within the visible spectrum.3. Chloride ions modulate this pH effect in a systematic and significant manner. For the pigment extracted in the chloride-deficient state the colour change occurs in the pH range of 6.0-7.0, the midpoint being close to 6.5, suggesting the possible participation of the imidazole group of histidine as the functional moiety. With added NaCl the colour shifts to the region below pH 6.2.4. The modulating action of chloride is postulated to be a conformational change of the opsin leading to a shift of the secondary interacting site from one functional group to another or else to a change in pK of a single group due to the conformational alteration of the electrostatics of the system.5. At pH values between 7.5 and 9.0 a different mechanism becomes apparent. In this region a decrease occurs in the photopigment density as well as a shift in absorbance toward the blue. This alkaline effect is readily reversed either by adding NaCl or else by lowering the pH. Along with the other protective effects of chloride these ions serve to reduce or prevent this alkaline loss in density.6. Associated with this reversible photopigment loss is a reversible appearance of a product with a maximum at about 366 nm. The spectrum of this product is like that produced by the addition of 11-cis retinal to the extract. Acidification of the alkaline preparation leads to a restitution of the photopigment as well as to a reduction of the 366-product.7. Addition of hydroxylamine to the alkaline extract in appropriate concentration inhibits the restitution of pigment-521 with acid or NaCl, but adding 11-cis retinal to the system leads to restoration of the photopigment after acidification. All the evidence suggests that product-366 is either free 11-cis retinal or else held to the opsin in a form that does not alter its spectral absorbance. The alkaline effect is therefore a disruption of the aldimine bond of the visual pigment.8. In many respects the gecko 521-pigment behaves like the chicken cone pigment, iodopsin, suggesting that an investigation of the latter in terms of pH may be a worthy project for future study.9. With its ability to change colour with pH, with chloride, with nitrate, etc. the extractable gecko pigment offers possibilities for the investigation of mechanisms responsible for adjusting visual pigment absorbance throughout the visible spectrum. The techniques of circular dichroism, Raman spectroscopy, infra-red spectroscopy, etc. may find here a suitable material for these studies.

Different reactivity of mitochondrial and cytoplasmic aspartate aminotransferases toward an affinity labeling reagent analog of the coenzyme.

The two isoenzymes of aspartate aminotransferase from pig heart have been reacted with a derivative of the coenzyme, 4'-N-(2,4-dinitro-5-fluorophenyl) pyridoxamine-5'-phosphate, which is a potential affinity labeling reagent. The derivative has a great affinity for both isoapoenzymes. In the cytosolic isoenzyme, the reversible binding is followed by a covalent labeling of the epsilon amino group of lysine 258, which usually forms an aldimine bond with pyridoxal-5'-phosphate. In the mitochondrial isoenzyme, no labeling occurs at the active site. The different reactivity indicates that a small but definite difference exists in the geometry of the two active sites. In the cytosolic isoenzyme also a sulfhydryl group outside the active site region, namely cysteine 45, reacts, but not by an affinity labeling mechanism. In both isoenzymes, the reversibly bound reagent slowly undergoes a splitting reaction by which pyridoxal-5'-phosphate is regenerated and activity re-established; the rate of this reaction is not fast enough to impaire the labeling potential of the reagent.

Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene Cross-link localization

At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the ϵ-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 Å (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5′-phosphate in the active site.

Conformational studies on the pyridoxal Schiff bases of polypeptides

AbstractThe pyridoxal Schiff bases of the polypeptides poly(L‐lysine), poly(L‐ornithine), and poly(L‐α,γ‐diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L‐α,γ‐diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.