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.     

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.     

Mechanism of O-acetylserine sulfhydrylase fromSalmonella typhimurium LT-2

Summary O-Acetylserine sulfhydrylase is a pyridoxal 5-phosphate (PLP) dependent enzyme that catalyzes the final step of L-cysteine biosynthesis inSalmonella, viz. the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. A spectrophotometric assay is available using 5-thio(2-nitrobenzoate) (TNB) as an analog of sulfide and monitoring the disappearance of absorbance at 412 nm. The enzyme catalyzes a ping pong mechanism with-aminoacrylate in Schiff base with the active site PLP as a covalent intermediate. Using data obtained from the pH dependence of kinetic parameters, the acid-base chemical mechanism and the optimum protonation state of enzyme and substrate functional groups necessary for binding has been determined. The Schiff base and the-amine of the substrate OAS are unprotonated for binding. There also appears to be a requirement for one active site general base to accept a proton from the-amine and to donate a proton to form cysteine. The enzyme also catalyzes an OAS hydrolase activity, and the pH dependence of this reaction suggests that the active site lysine that participated in the Schiff base linkage is protonated to start the second half reaction, and has a pK of about 8.2. The stereochemistry of³H-borohydride reduction of the Schiff base in free enzyme has been determined by degradation of the resulting pyridoxyllysine to pyridoxamine and measuring³H-release with apo-aspartate aminotransferase. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.     

Modulation of Cystathionine β-Synthase Activity by the Arg-51 and Arg-224 Mutations

Human cystathionine β-synthase (CBS) catalyzes a pyridoxal 5′-phosphate (PLP) dependent β-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 Å away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate

The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit.Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.     

Formation of -cyanoalanine by O-acetylserine sulfhydrylase

Cell-free extracts of Bacillus megaterium form beta-cyanoalanine (beta-CNA)-(14)C from Na(14)CN and l-cysteine, O-acetyl-l-serine or, to a lesser extent, l-serine. However, the presence of cyanide in the growth medium does not increase the capacity of cell extracts to catalyze the formation of beta-CNA from cysteine and cyanide. The formation of beta-CNA is readily detected in extracts of cells grown in synthetic media with sulfate or l-djenkolic acid as sulfur sources; such cells also exhibit an increased ability to form cysteine when compared with cells grown on cysteine as the sulfur source. beta-CNA formation could not be detected in extracts of cells grown on cysteine as the sulfur source. A 40-fold purification of the O-acetyl-serine sulfhydrylase resulted in the co-purification of the beta-CNA-forming activity. The sulfhydrylase and the beta-CNA-forming activity co-chromatographed on diethyl-aminoethyl cellulose and Sephadex G-100.     

Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in l-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0A resolution

O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-wavelength anomalous dispersion. A monomer consists of three domains, including an N-terminal domain with a new alpha/beta fold. The topology folds of the middle and C-terminal domains were similar to those of the O-acetylserine sulfhydrylase-A from Salmonella typhimurium and the cystathionine beta-synthase from human. The cofactor, pyridoxal 5'-phosphate, is bound in a cleft between the middle and C-terminal domains through a covalent linkage to Lys127. Based on the structure determined, O-phospho-l-serine could be rationally modeled into the active site of the enzyme. An enzyme-substrate complex model and a mutation experiment revealed that Arg297, unique to hyperthermophilic archaea, is one of the most crucial residues for O-phosphoserine sulfhydrylation activity. There are more hydrophobic areas and less electric charges at the dimer interface, compared to the S.typhimurium O-acetylserine sulfhydrylase.     

O-acetylserine sulfhydrylase and S-sulfocysteine synthase activities of Chromatium vinosum

Two proteins containing O-acetylserine sulfhydrylase activity were purified from Chromatium vinosum. Their separation was carried out by DE52 or Ecteola cellulose chromatography. While protein I with a molecular weight of 56,000 had only O-acetylserine sulfhydrylase activity, protein II with a molecular weight of 50,000 possessed S-sulfocysteine synthase activity in addition. It was not possible to separate the two activities of protein II by electrophoretic methods. The reaction rate of protein II with sulfide and O-acetylserine was twice as high as that with thiosulfate and O-acetylserine. When extracts of sulfate-grown cells were purified the major O-acetylserine activity was always associated with protein II. Regulatory and kinetic phenomena of the two activities were studied.     

Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase

Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

Role of Histidine-152 in cofactor orientation in the PLP-dependent O-acetylserine sulfhydrylase reaction

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of l-cysteine, the replacement of the β-acetoxy group of O-acetyl-l-serine (OAS) by a thiol. The 5′-phosphate of the PLP cofactor is very tightly bound to the enzyme; it accepts 8 hydrogen bonds from enzyme side chains and a pair of water molecules, and is in close proximity to a helix dipole. Histidine-152 (H152) is one of the residues that, via a water molecule, is responsible for positioning the 5′-phosphate. Mutation of H152 to alanine was predicted to increase the freedom of the 5′-phosphate, and as a result the cofactor, giving a decrease in the overall rate of the reaction. The H152A mutant enzyme was thus prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and ³¹P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. UV-visible absorbance and visible CD spectra are consistent with a shift in the ketoeneamine to enolimine tautomeric equilibrium toward the neutral enolimine in the internal Schiff base of the free enzyme (ISB), the amino acid external Schiff base (ESB), and the α-aminoacrylate intermediate (AA). ³¹P NMR spectra clearly indicate the presence of two conformers (presumably open and closed forms of the enzyme) that interconvert slowly on the NMR time scale in the ISB and ESB. Kinetic data suggest the decreased rate of the enzyme likely reflects a decrease in the amount of active enzyme as a result of an increased flexibility of the cofactor which results in substantial nonproductive binding of OAS in its external Schiff base, and a stabilization of the external Schiff bases of OAS and S-carboxynitrophenyl-l-cysteine. The nonproductive binding and stabilization of the external Schiff bases are thus linked to the shift in the tautomeric equilibrium and increase in the rate of interconversion of the open and closed forms of the enzyme. The location of the 5′-phosphate in the cofactor-binding site determines additional interactions between the cofactor and enzyme in the closed (ESB) form of the enzyme, consistent with an increased rate of interconversion of the open and closed forms of the enzyme upon increasing the rate of flexibility of the cofactor.     

Evidence for O-acetylserine in Nicotiana tabacum

O-Acetylserine, a precursor of cysteine in plants, was isolated from cells of Nicotiana tabacum cultured in liquid medium.     

Refining the structure−activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms

The lack of efficacy of current antibacterials to treat multidrug resistant bacteria poses a life-threatening alarm. In order to develop enhancers of the antibacterial activity, we carried out a medicinal chemistry campaign aiming to develop inhibitors of enzymes that synthesise cysteine and belong to the reductive sulphur assimilation pathway, absent in mammals. Previous studies have provided a novel series of inhibitors for O-acetylsulfhydrylase – a key enzyme involved in cysteine biosynthesis. Despite displaying nanomolar affinity, the most active representative of the series was not able to interfere with bacterial growth, likely due to poor permeability. Therefore, we rationally modified the structure of the hit compound with the aim of promoting their passage through the outer cell membrane porins. The new series was evaluated on the recombinant enzyme from Salmonella enterica serovar Typhimurium, with several compounds able to keep nanomolar binding affinity despite the extent of chemical manipulation.     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.     

Purification and Properties of the Trypsin Inhibitors from Buckwheat Seed

The trypsin inhibitors in buckwheat seeds were isolated by affinity chromatography on trypsin-Sepharose 4B, and the components were fractionated by chromatography on DEAE-Sepharose CL-6B. The major components, inhibitors I, II and III, were found to be homogeneous proteins with molecular weight of about 8,000. Trypsin inhibitory activity was more pronounced than the chymotrypsin inhibitory activity in all the inhibitor preparation obtained. The three major inhibitors had similar amino acid compositions and had no detectable amounts of tryptophan and carbohydrate. A high level of acidic and basic amino acid residues and a low level of methionine, tyrosine and phenylalanine residues characterized the inhibitors. Although the inhibitors I and II were particularly thermostable, inhibitor III, the most abundant component, was shown to be relatively heat-labile.