Moles Of Pyridoxal Phosphate Research Articles

O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Further purification and characterization as a pyridoxal enzyme.

O-Acetylserine-O-acetylhomoserine sulfhydrylase [EC class 4.2.99], catalyzing the sulfhydrylation of both O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) (O-acetyl-L-serine(O-acetyl-L-homoserine) + H2S leads to L-cysteine (L-homocysteine) + acetate), was extracted and purified from bakers' yeast by an improved method. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecylsulfate and by ultracentrifugal analysis. The apo-enzyme was protected by pyridoxal phosphate (PALP) from inactivation by heat, urea, and trypsin [EC 3.4.21.4], suggesting that the binding of PALP to the apo-enzyme rendered the conformation of the protein more stable. The holo-enzyme showed absorption peaks at 420 and 330 nm due to bound PALP, in addition to a peak at 280 nm. Upon reduction with borohydride, the 420-nm peak disappeared and an increase in the 330-nm peak occurred concomitant with loss of the catalytic activity. Lysine appeared to be the pyridoxal binding site, based on identification of pyridoxyl-lysine in the hydrolyzate of the holo-enzyme. It was shown by both spectral and chemical determinations that 4 moles of PALP could bind to 200,000 g of apo-protein. The apo-enzyme showed a lower association constant with PALP than some other enzymes. Pyridoxal inhibited the activity competitively with respect to PALP. Based on these findings, it appears that the reaction mechanism of this enzyme is similar to those of other pyridoxal enzymes.

Purification and properties of S-alkyl-L-cysteine lyase from seedlings of Acacia farnesiana Willd.

1. An S-alkyl-L-cysteine lyase (EC 4.4.1.6) was purified to apparent homogeneity from extracts of acetone-dried powders of the hypocotyls of etiolated 5-day-old seedlings of Acacia farnesiana Willd. 2. The enzyme catalyses a beta-elimination reaction and will utilize both the thioether and sulphoxide form of the substrate. 3. There is a braod specificity with regard to the alkyl substituent, but cystathionine is utilized very poorly. 4. The pH optimum is 7.8 and the Km value for the probable natural substrate L-djenkolate is 0.3 mM. 5. Both sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ultracentirfugal analysis give a molecular weight of about 144000. 6. One mol of pyridoxal phosphate is bound/mol of enzyme. 7. The energy of activation with L-djenkolate as the substrate is 53.1 kJ/mol. 8. The enzyme has a partial specific volume of 0.56 and S20,w 7.26S.

Effet du phosphate de pyridoxal sur les triptophanases de cinq espèces d'Entérobactériaceae

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.

Cysteine synthase from rape leaves.

Cysteine synthase [O-Acetyl-L-serine acetate-lyase (adding hydrogen-sulfide) EC 4.2.99.8] has been highly purified from the extract of rape, Brassica chinensis var. Komatsuna. The purified preparation appeared to be homogeneous on Sephadex G-100 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis, showing a molecular weight of about 62,000. The latter method also suggested that this enzyme was composed of two identical subunits. The enzyme contained 2 moles of pyridoxal phosphate per mole of enzyme.

Active-sitve titration of pig plasma benzylamine oxidase with phenylhydrazine.

Pig plasma benzylamine oxidase is a protein containing cupric copper and pyridoxal phosphate. The pyridoxal phosphate is stably linked to the enzyme. Discrepancies in the numbers of active sites per molecule of enzyme are reported in the literature. This paper shows that the fully active pure enzyme contains 3 mol of pyridoxal phosphate per mol, whereas enzymes with a lower specific activity are shown by titration with phenylhydrazine to have a lower pyrdoxal phosphate content.

Studies on a 2,3-Diaminopropionate:Ammonia-Lyase from a Pseudomonad

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.

Interactions of 5′-Adenosine Diphosphate and l-Threonine with the Quaternary Structure of l-Threonine Dehydratase: I. NATURE OF THE ENZYMIC SUBSTRUCTURE

Abstract An analysis of the subunit structure of l-threonine dehydratase (l-threonine hydro-lyase (deaminating)) from Clostridium tetanomorphum has revealed that the native enzyme, with a molecular weight of 184,000, is composed of four apparently identical subunits. This conclusion was based on the results obtained from peptide maps, cyanogen bromide cleavage patterns, sedimentation coefficients of maleylated enzyme, and molecular weight estimated in 6 m guanidine hydrochloride plus 1 mm mercaptoethanol. Furthermore, threonine dehydratase was found to contain 4 moles of pyridoxal phosphate per mole of native enzyme. Evidence was also gathered that the protomer of molecular weight 46,000 was composed of two nonidentical peptide chains of approximately 29,000 and 17,000 molecular weight. This was supported by sodium dodecyl sulfate gel electrophoresis, carboxyl-terminal analysis, chromatography on guanidine-agarose columns, and sedimentation equilibrium ultracentrifugation of carboxamidomethylated enzyme in 6 m guanidine hydrochloride. The fact that strongly dissociating and reducing conditions were necessary to produce species less than 46,000 suggest that the subprotomeric polypeptide chains are disulfide bridged.

Paradoxical inhibition of phosphorylase by pyridoxal phosphate. 3. Sequence of amino acids at site of reaction.

Pyridoxal phosphate phosphorylase b prepared from native phosphorylase b by the specific incorporation of 1–2 mol of pyridoxal phosphate (PLP) differs from the native enzyme in its absorbance, fluorescence, and kinetic properties. The secondary site of attachment of the PLP group responsible for these changes has been identified as a lysine residue from the formation of PLP-lysine on NaBH4 reduction. The peptide containing this derivative was isolated from a pepsin digest of the modified protein by Sephadex G-25 and Chromobeads type P resin column chromatography, followed by rechromatography on the resin after removal of the phosphate by alkaline phosphatase. The amino acid sequence of the purified hexapeptide was determined to be Tyr–(Pxy)Lys–Asn–Pro–Arg–Glu (Pxy, pyridoxyl). A second lysine reacting with PLP was found in the sequence Phe–Glu–Gly-(Pxy)Lys–Glu.

The Purification and Subunit Structure of Cysteine Desulfhydrase from Salmonella typhimurium

Abstract The inducible enzyme, cysteine desulfhydrase, was purified from Salmonella typhimurium to a state of near homogeneity. This enzyme has a very low tryptophanase activity, lacks cystathionase and tryptophan synthetase activities, and appears to be a specific cysteine desulfhydrase. The purified native enzyme has an s20,w of 10.4 and a molecular weight of 229,000 as determined by equilibrium sedimentation. Polyacrylamide electrophoresis in sodium dodecyl sulfate showed a single species of polypeptide chain with a molecular weight of 37,000 while the pyridoxal phosphate content was found to be 1 mole per 37,700 g of protein. These data, together with the results of amino acid analyses, tryptic peptide maps, and an NH2-terminal amino acid analysis, indicate that the native enzyme is a hexamer, containing 6 moles of pyridoxal phosphate, and composed of six identical polypeptide chains with NH2-terminal serines.

Characterization of a previously unidentified hemoglobin fraction

The hemoglobin peaks designated as “unnamed”, Hb 1a+b, Hb A 1c, Hb A GSSG, Hb A 0, and Hb A 2 were separated from human red cell hemolysate by CM-Sephadex chromatography. The first peak of Hb, unnamed, was found to have 1 mole of pyridoxal phosphate per mole of Hb tetramer. Treatment of hemolysate with pyridoxal phosphate increased the total amount of Hb in this peak without changing the ratio of pyridoxal phosphate to Hb. Substantial amounts of pyridoxal phosphate were not found in any other Hb fraction. Cord blood hemolysate, consisting mainly of Hb F is pyridoxylated to a much smaller extent than adult hemoglobin.

L-Threonine Deaminase of Rhodospirillum rubrum. Purification and Characterization

A procedure for the purification of threonine deaminase from Rhodospirillum rubrum is described. The 2200-fold purified enzyme was judged to be pure by disc-gel electrophoresis and ultracentrifugation. Sedimentation velocity centrifugation yielded an s:,,~ value of 8.1 S and a D~O,~ of 4.65 x 10-7 em2 x see-l. Assuming a partial specific volume of 0.74 g/ml the molecular weight of 164000 was calculated from the Svedberg equation. However, from calibrated Sephadex G-200 columns, a D~O,~ of 4.15 x 10-7 em2 x sec-l was obtained and using this equation, the molecular weight was calculated to be 180000. Electrophoresis of the enzyme on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the native enzyme was composed of four subunits, each of about 46000 molecular weight. In addition to the native tetrameric molecule, an active octameric species was detected in polyacrylamide gel electrophoresis. The spectrum of the pure enzyme showed absorption maxima at 279nm and 412nm, characteristic of pyridoxal 5’-phosphate-containing enzymes. Analysis of the cofactor content revealed the presence of 4.2 moles of pyridoxal phosphate per 180000 g of protein. The enzyme displayed a normal Michaelis-Menten substrate saturation curve in the absence of isoleucine. High concentrations of isoleucine inhibited the enzyme activity somewhat only at low levels of the substrate ; the inhibition was weak as compared to all other biosynthetic threonine deaminases. No other amino acid or any nucleotide tested produced any significant activation or inhibition. In assays at pH values greater than 8.4, a transient build up of a 245 nm absorbing species was observed. It is suggested that this species corresponds to one of the two intermediates of the deamination reaction, a-aminocrotonate or a-iminobutyrate, and that at high pH the ratelimiting step in the reaction is the hydrolysis of the intermediate to the final products, 2-0x0butyrate and ammonia.

The B Protein of Escherichia coli Tryptophan Synthetase: I. EFFECTS OF SULFHYDRYL MODIFICATION ON ENZYMATIC ACTIVITIES AND SUBUNIT INTERACTION

Abstract Sulfhydryl modification of the B protein of Escherichia coli tryptophan synthetase has been carried out to investigate the role of sulfhydryl residues in the active center of this protein and in its interaction with the A protein. Both N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with two sulfhydryls per monomer of apo-B protein and one sulfhydryl per monomer of the holo-B protein, which contains 1 mole of pyridoxal phosphate per monomer. The serine deaminase activity and the tryptophan synthetase activity are both largely lost after the reaction of one sulfhydryl in the holo-B protein with DTNB. Similar results are obtained after the reaction of one sulfhydryl in the apo-B protein with NEM. In contrast, after the reaction of one sulfhydryl in the holo-B protein with NEM, the serine deaminase activity is somewhat enhanced, whereas, the tryptophan synthetase activity is largely lost. Kinetic comparisons of the NEM-modified holo-B protein with the untreated holo-B protein show that the Km for l-serine is largely unchanged, but that both the Km for indole and the Vmax in the tryptophan synthetase reaction are changed by modification. Since one sulfhydryl in the B protein is fully protected from NEM modification by pyridoxal phosphate and a second sulfhydryl is partially protected from modification in the holo-B protein by the substrate l-serine, both of these sulfhydryl residues may be located in the active center of the protein. The reactive sulfhydryl residue in the holo-B protein is also protected by interaction with the A protein. However, this sulfhydryl is not necessary for the interaction of the A and B proteins since the holo-B protein modified with 1 mole of NEM still associates normally with the A protein and facilitates its hydrolysis of indole-3-glycerol phosphate.

Crystalline threonine aldolase from Candida humicola

The preparation of crystalline threonine aldolase from the cell extract of Candida humicola is described. The enzyme is homogeneous upon ultracentrifugation and disc electrophoresis. The molecular weight is 277,000. The enzyme exhibits an absorption maximum at 420 mμ, and contains 6 moles of pyridoxal phosphate per mole of enzyme. The enzyme catalyzes the stoichiometric conversion of L-threonine into acetaldehyde and glycine. The enzyme also catalyzes the glycine formation from D, L-allothreonine and L-serine.

Purification and properties of serine hydroxymethylase from soluble and mitochondrial fractions of rabbit liver

Serine hydroxymethylase ( l-serine tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1) isoenzymes were purified separately from the soluble and mitochondrial fractions of rabbit liver. The purified preparations appeared to contain a single component in each case as judged from ultracentrifugation and eletrophoresis on polyacrylamide or starch gels. The isoenzymes had similar properties with respect to pH optimum, electrophoretic mobility as well as the reactions they catalyzed. The isoenzymes, however, were different immunochemically; the antiglobulin prepared against one of the isoenzymes did not inhibit the enzymic activity of the other. The molecular weights of the soluble fraction and the mitochondrial fraction enzymes, determined by the sedimentation equilibrium method, were approx. 185 000 and 170 000, respectively. Both isoenzymes contained 4 moles of pyridoxal phosphate per mole of enzyme.

Studies on the Interaction between Regulatory Enzymes and Effectors: III. Crystallization and characterization of adenosine 5′-monophosphate-dependent threonine deaminase from escherichia coli

Abstract Biodegradative l-threonine deaminase from Escherichia coli was isolated and crystallized in the presence of AMP. The enzyme was homogeneous as judged by ultracentrifugation and starch gel electrophoresis. The molecular weight was estimated to be approximately 147,000, and the amino acid composition was determined. The enzyme had a marked yellow color with an absorption maximum at 415 mµ and contained 4 moles of pyridoxal phosphate per mole of protein. It was found that the crystalline enzyme binds 4 moles of AMP per mole of enzyme.

Threonine Deaminase from Salmonella typhimurium: I. PURIFICATION AND PROPERTIES

Abstract Biosynthetic l-threonine deaminase (l-threonine hydrolyase, deaminating; EC 4.2.1.16) has been purified approximately 250-fold from crude extracts of nutritionally derepressed Salmonella typhimurium. Sedimentation of the enzyme in the ultracentrifuge results in a single, sharp, symmetrical peak in the schlieren pattern. The purification procedure is capable of removing the radioactivity from a crude extract containing label in all the proteins except native threonine deaminase. The deaminase reaction shows homotropic interaction only in the presence of l-isoleucine; high ionic strength or low valine concentrations remove these interactions. The pure enzyme is effectively inhibited by l-isoleucine. The pH optimum of the deaminase reaction is 9 to 10; the pH curve is shifted to higher values in the presence of l-isoleucine. The activity of the purified enzyme is independent of added pyridoxal phosphate. The cofactor is resolved by dialysis against tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 8.0. Pyridoxal phosphate and pyridoxamine phosphate are capable of reactivating the enzyme. The activity of the purified enzyme is independent of divalent cations, but NH4+, K+, Li+, and Na+ show about 20% stimulation of enzyme activity. The enzyme has two absorption maxima (280 and 420 mµ). The removal of pyridoxal phosphate from the enzyme is accompanied by the loss of the 420 mµ absorption. The enzyme contains 0.99 mole of pyridoxal phosphate per 100,000 g of protein. The minimum molecular weight computed on the basis of pyridoxal phosphate content and sedimentation coefficient is 200,000.

Reactivating effect of some naturally occurring dithiol compounds on threonine deaminase

The biodegradative threonine deaminases have been purified to homogeneity and crystallized from the extracts of Escherichia coli (E. coli) and Clostridium tetranomorphum, respectively. It has been demonstrated that deaminases are composed of four identical subunits, each monomer containing one mole of pyridoxal phosphate. The deamination reaction mechanism studied using the E. coli enzyme is compatible with the mechanism derived from the pyridoxal-catalyzed α,β-elimination reaction in the non-enzymic model system, although direct evidence for the participation of pyridoxal phosphate in the enzyme reaction is lacking. Spectral and circular dichroic changes of the enzyme-bound pyridoxal phosphate after addition of the substrate, L-threonine, were postulated to represent the formation of a complex between pyridoxal phosphate and the dehydrated reaction intermediate. Optical studies indicate that optical changes occur prior to the β-elimination reaction. Kinetic analyses have indicated that the enzyme exists as an inactive form under the conditions of these optical experiments. These phenomena could be interpreted as indicating that the conversion of this inactive enzyme into an active form occurs after addition of L-threonine. These optical changes are observed during the reaction and this conversion step appears to be the rate-limiting step of the overall reaction under the conditions employed.

Tyrosine Aminotransferase: PURIFICATION AND CHARACTERIZATION

Abstract Tyrosine aminotransferase was highly purified from rat livers in which the enzyme had been induced by a glucocorticoid hormone. The preparation eluted from a DEAE-cellulose column as a single peak gave a single precipitin line by the Ouchterlony double diffusion precipitin reaction and sedimented as a single boundary in the ultracentrifuge with an s20,w of 5.9. A molecular weight of 91,000 was determined by equilibrium sedimentation. Despite these indications of homogeneity, three enzymatically active bands were observed in disk-gel electrophoresis. The purified enzyme is yellow in solution and exhibits absorption maxima at 425 mµ and 327 mµ (at slightly acidic pH), which indicate an interaction of the enzyme with its cofactor, pyridoxal phosphate. Addition of tyrosine shifted the 425 mµ peak to 327 mµ, whereas α-ketoglutarate, the keto-acid substrate, reversed this shift. Separation of enzyme from coenzyme could be achieved by dialyzing holoenzyme with tyrosine in 0.1 m phosphate buffer. Addition of pyridoxal phosphate to apoenzyme caused return of the 425 mµ peak and full reconstitution of activity. One mole of pyridoxal phosphate is bound per 94,000 g of enzyme. Tyrosine aminotransferase has a bell-shaped pH-activity curve with a maximum near pH 7.5. The following apparent Michaelis constants were determined: tyrosine, 1.7 x 10-3 m, α-ketoglutarate, 7 x 10-4 m, pyridoxal phosphate, 1.7 x 10-8 m, and pyridoxamine phosphate, 1.5 x 10-7 m. Phosphate was shown to competitively inhibit the binding of the two cofactors to enzyme with Ki values of 1.1 x 10-3 m and 3.4 x 10-3 m for pyridoxal phosphate and pyridoxamine phosphate, respectively.

Glycine Metabolism: IV. EFFECT OF BOROHYDRIDE REDUCTION ON THE PYRIDOXAL PHOSPHATE-CONTAINING GLYCINE DECARBOXYLASE FROM PEPTOCOCCUS GLYCINOPHILUS

The pyridoxal phosphate-containing glycine decarboxylase from Peptococcus glycinophilus exhibited fluorescence emission peaks at 390 and 500 mµ when activated at 340 and 420 mµ, respectively. Removal of the pyridoxal phosphate resulted in the loss of the 500 mµ peak, but did not affect the 390 mµ peak. When the holoenzyme was reduced with sodium borohydride only a single emission peak at 390 mµ was detectable, but the intensity of the emission was approximately 10-fold greater than the emission at 390 mµ of the nonreduced holoenzyme or of either the reduced or nonreduced apoenzyme. Estimation of the molar ratio of pyridoxal phosphate to enzyme protein was achieved by comparing the increase in fluorescence at 390 mµ, the increase in ultraviolet absorption at 430 mµ, and the increase in catalytic activity of a known amount of apoenzyme upon incubation with increasing amounts of pyridoxal phosphate. It was found that each mole of enzyme bound 2 moles of pyridoxal phosphate.

PYRIDOXAL PHOSPHATE AS A CONSTITUENT OF THE HISTAMINASE (BENZYLAMINE OXIDASE) OF PIG PLASMA.

The histaminase (‘benzylamine oxidase’) of pig plasma has recently been crystallized and a description is given of some of the physical and chemical properties of the pure preparation of the oxidase. The fluorescence of the enzyme is typical of a protein containing tryptophan. The phosphorus content is about four atoms per molecule. On enzymic hydrolysis a product has been obtained that has the fluorescence properties of a pyridoxal protein. This product gave rise on acid hydrolysis to material capable of activating the L-tyrosine apodecarboxylase of Streptococcus faecalis R in the presence of adenosine triphosphate, indicating that pyridoxal was set free in the hydrolysis. Acid hydrolysis of a solution of the crystalline enzyme and subsequent treatment with urea yielded diffusible material with the spectroscopic and fluorescence properties of pyridoxal. Using the bacterial apodecarboxylase, traces of pyridoxal phosphate and larger amounts of free pyridoxal were shown to be present. It was calculated that about 3 or 4 moles of pyridoxal were present per mole of enzyme. Together with the phosphate estimations, these observations lead to the conclusion that the oxidase contains 3 or 4 moles of pyridoxal phosphate per mole. Observations on rats maintained on a diet deficient in vitamin B 6 are in agreement with these conclusions.