Ionization state of pyridoxal 5'-phosphate in D-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by 31P NMR spectroscopy

The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.     

A reaction pathway for transimination of the pyridoxal 5'-phosphate in D-serine dehydratase by amino acids

Transimination of the enzyme-linked cofactor by an amino acid is the first chemical transformation in the reactions catalyzed by pyridoxal 5'-phosphate-requiring enzymes. In this work, stopped flow fluorimetry was used to characterize the kinetics of transimination of the cofactor in D-serine dehydratase by several amino acids. The results of these studies indicate that transimination is a multistep process, the first step of which is probably formation of a noncovalent complex between the enzyme and the amino acid. D-Serine dehydratase was found to exhibit considerable specificity in the transimination reaction. Furthermore, the enzyme was shown to facilitate the transimination reaction with amino acids and inhibit transimination of the bound cofactor by amines lacking a carboxylate group. A reaction pathway was proposed for the transimination process which accounts for the specificity of the enzyme and indicates the changes in the conformation of the bound cofactor as dictated by the stereoelectronic requirements of the transimination reaction.     

(31)P NMR spectroscopy senses the microenvironment of the 5'-phosphate group of enzyme-bound pyridoxal 5'-phosphate

In this review it is demonstrated that (31)P NMR spectroscopy can be used to elucidate information about the microenvironment around the phosphate group of enzyme-bound pyridoxal 5'-phosphate (PLP). The following information can be obtained for all PLP-dependent enzymes: 1) the protonation state of the 5'-phosphate and its exposure to solvent, and 2) tightness of binding of the 5'-phosphate. In addition, the 5-phosphate can report on the protonation state of the Schiff base lysine in some enzymes. Changes in the 5'-phosphate chemical shift can be used to determine changes in tightness of binding of the phosphate as the reaction pathway is traversed, providing information on the dynamics of the enzyme. (31)P NMR spectroscopy is thus an important probe of structure, dynamics and mechanism in native and site-directed mutations of PLP-dependent enzymes. Examples of all of the above are provided in this review. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis

D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue).     

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.     

Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.     

Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.     

Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site.

The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.     

Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C.     

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes.

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C alpha and C-4' positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular pKa of the monoprotic base. Thus the epsilon-ammonium group of the internal aldimine-forming lysine residue operates at C-4'-si-face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C alpha from the 4'-si-face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldimination processes and as a binding group for the alpha-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. -S-X-H-K-) with that for aspartate aminotransferase (e.g. -S-X-A-K-) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of quinonoid species from pyridoxal 5'-phosphate

To establish the state of protonation of quinonoid species formed nonenzymically from pyridoxal phosphate (PLP) and diethyl aminomalonate, we have studied absorption spectra of the rapidly established steady-state mixture of species. We have evaluated the formation constant and the spectrum of the mixture of Schiff base and quinonoid species. For N-methyl-PLP a singly protonated species with a peak at 464 nm is formed from the unprotonated aldehyde and the conjugate acid of diethyl aminomalonate with a formation constant Kf of 240 M-1. The very intense absorption band with characteristic vibrational structure (most evident as a shoulder at 435 nm) is accompanied by a weaker, structured band at about 380 nm and a weak, broad band at 330 nm. We suggest that the 380-nm band may represent a tautomeric form of the quinonoid compound. Protonation of the phosphate group appears to affect the spectrum only slightly. The corresponding mixture of Schiff base and quinonoid species formed from PLP has a very similar spectrum at pH 6-7. It has a formation constant Kf of 230 M-1 and a pKa of 7.8, which must be attributed to the ring nitrogen atom. The dissociated species, which may be largely carbanionic, has a strong structured absorption band at 430 nm and a weaker one, again possibly a tautomer, in the 330-nm region. The analysis establishes that in all species a proton remains on either the phenolic oxygen or the imine nitrogen. Proton NMR spectroscopy, under some conditions, reveals only two components: free PLP and what appears to be Schiff base. However, we suggest that the latter may, in fact, be a quinonoid form, either alone or in rapid equilibrium with the Schiff base. Absorption spectra of quinonoid species formed in enzymes are analyzed and compared with the spectra of the nonenzymic species.     

The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.

Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1. The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium. The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration. The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

Structural role of pyridoxal 5'-phosphate, pyridoxal 5'-phosphate analogs, and other agents in the association of subunits of Bacillus alvei apotryptophanase.

Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.     

Critical hydrogen bonds and protonation states of pyridoxal 5'-phosphate revealed by NMR

In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5'-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from (15)N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid-base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.