Chemogenomics of pyridoxal 5′-phosphate dependent enzymes

Pyridoxal 5′-phosphate (PLP) dependent enzymes comprise a large family that plays key roles in amino acid metabolism and are acquiring an increasing interest as drug targets. For the identification of compounds inhibiting PLP-dependent enzymes, a chemogenomics-based approach has been adopted in this work. Chemogenomics exploits the information coded in sequences and three-dimensional structures to define pharmacophore models. The analysis was carried out on a dataset of 65 high-resolution PLP-dependent enzyme structures, including representative members of four-fold types. Evolutionarily conserved residues relevant to coenzyme or substrate binding were identified on the basis of sequence-structure comparisons. A dataset was obtained containing the information on conserved residues at substrate and coenzyme binding site for each representative PLP-dependent enzyme. By linking coenzyme and substrate pharmacophores, bifunctional pharmacophores were generated that will constitute the basis for future development of small inhibitors targeting specific PLP-dependent enzymes.     

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.     

Reactivity of the phosphopyridoxal groups of cystathionase.

Aminooxyacetate and alpha-amino-gamma-aminooxybutyrate (canaline) react specifically with the P-pyridoxal groups of cystathionase to produce characteristic changes in the absorption and fluorescence properties of the bound cofactor. The increase in fluorescence at 450 nm was used to monitor the reaction. Aminooxyacetate attacks the Schiff base linkage of the enzyme several times faster (k1 = 3700 M-1 min-1 and k2 = 1000 M-1 min-1) than it attacks the aldehydic carbon of free P-pyridoxal (k = 290 M-1 min-1). Similar results were obtained with canaline. The kinetic studies indicate that a Schiff base linkage in the enzyme cystathionase should offer direct kinetic advantage during the reaction between the substrate and the cofactor. It is also shown that the inhibitor L-alpha-gamma-aminobutyrate reacts with bound P-pyridoxal to form free P-pyridoxamine. The rate of formation of P-pyridoxamine parallels the rate of enzyme inactivation.     

Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate

Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.     

Brain pyridoxine-5-phosphate oxidase. Modulation of its catalytic activity by reaction with pyridoxal 5-phosphate and analogs

Pyridoxine-5-P oxidase, the flavoprotein involved in the oxidation of pyridoxamine-5-P and pyridoxine-5-P to pyridoxal-5-P, has been isolated and purified to homogeneity using sheep brain tissues. Inactivation of the oxidase by bis-pyridoxal-5-P results in binding of the inhibitor to specific lysyl residues. After NaBH4 reduction of the inactivated enzyme, it was found that 1 P-pyridoxyl-pyridoxine-P residue was incorporated per enzyme dimer. After trypsin digestion of the bis-PLP modified enzyme, only one peptide absorbing at 320 nm, was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the labeled peptide was determined by automated Edman degradation. The observations reported in this paper are relevant to the mechanisms underlying the regulation of the catalytic function of pyridoxines-5-P oxidase by the product pyridoxal-5-P. It is postulated that the catalytic function of the oxidase is modulated by binding of pyridoxal-5-P to a specific lysyl residue of the dimeric structure of the protein.     

A new fluorometric method for the determination of pyridoxal 5′-phosphate

A new fluorometric method using semicarbazide for the determination of pyridoxal and pyridoxal 5′-phosphate (PLP) in whole blood, red cells and plasma has been developed. Semicarbazide breaks the Schiff base of PLP and proteins by “trans-Schiffization” reaction and forms semicarbazone of PLP. The semicarbazone of PLP emits strongly at 460 nm when excited at 380 nm. Several metabolic intermediates were tested for the possible interference. Only pyridoxal was found to interfere. The interference can be corrected since pyridoxal emits at 380 nm when excited at 320 nm. Using this method we found that rabbit red cells in vivo are freely permeable to PLP.     

Observation of nanosecond transient states in laser-induced reactions of pyridoxal 5′-phosphate

A nanosecond laser-flash photolysis system is described. The intensity of the 5-ns resolved pulses of the analyzing flash can be digitized with 0.03% precision. As a result, the electronic absorption spectra of transients in 60-μl samples of 10^?4m enzyme have been determined.     

4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate

Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.     

A convenient lactic dehydrogenase-coupled assay for determining pyridoxal 5′-phosphate in plasma

An assay for determining the concentration of pyridoxal 5′-phosphate in plasma from 0.4 ml whole blood is reported. The assay consists of incubating deproteinized plasma with d-serine apodehydratase from Escherichia coli in 0.5 mN-2-hydroxyethyl-piperazine-N′-3-propanesulfonic acid, pH 7.8, at 37°C for 15 min, and then determining the d-serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25°C) was used to measure the rate of enzymically catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal 5′-phosphate in the plasma sample was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5′-phosphate present in the assay.     

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate

The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP. In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used. The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate. Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated. In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP. The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed. The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate.     

Preparation and characterization of several new immobilized derivatives of pyridoxal 5′-phosphate

Two types of new Sepharose-bound pyridoxal 5′-phosphate, N-immobilized and 3-0-immobilized pyridoxal 5′-phosphate analogues, were prepared by reacting pyridoxal 5′-phosphate with a bromoacetyl derivative of Sepharose 4B in dimethylformamide (50% v/v) and in potassium phosphate buffer (pH 6.0) for approx. 70 h at room temperature in the dark, respectively. The properties of these immobilized pyridoxal 5′-phosphate derivatives including their catalytic activities in the non-enzymatic cleavage reaction of tryptophan were studied in comparison with those of the 6-immobilized pyridoxal 5′-phosphate analogue reported previously by the present authors. The usefulness of these pyridoxal 5′-phosphate analogues in the preparation of immobilized tryptophanase was demonstrated.     

Rapid purification by affinity chromatography of rat brain pyridoxal kinase and pyridoxamine-5-phosphate oxidase

Rat brain pyridoxal kinase and pyridoxamine 5′ phosphate oxidase have been purified to electrophoretic homogeneity using pyridoxyl Sepharose and phosphopyridoxyl Sepharose columns as the first stages in the purification procedures. These affinity supports were synthesized by two simple steps consisting of reacting pyridoxal and pyridoxal 5′ phosphate with commercially available ω-aminohexyl Sepharose and subsequent reduction of the resultant Schiff's bases with sodium borohydride. This method allows total purification of both enzymes from the same tissue source in 4–5 days.     

Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1

In most eubacteria, fungi, apicomplexa, plants and some metazoans, the active form of vitamin B6, PLP (pyridoxal 5-phosphate), is de novo synthesized from three substrates, R5P (ribose 5-phosphate), DHAP (dihydroxyacetone phosphate) and ammonia hydrolysed from glutamine by a complexed glutaminase. Of the three active sites of DXP (deoxyxylulose 5-phosphate)independent PLP synthase (Pdx1), the R5P isomerization site has been assigned, but the sites for DHAP isomerization and PLP formation remain unknown. In the present study, we present the crystal structures of yeast Pdx1/Snz1, in apo-, G3P (glyceraldehyde 3-phosphate)- and PLP-bound forms, at 2.3, 1.8 and 2.2 ? (1 ?=0.1 nm) respectively. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfer site. We propose a putative catalytic mechanism for Pdx1/Snz1 in which R5P and DHAP are isomerized at two distinct sites and transferred along well-defined routes to a final destination for PLP synthesis.