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PubMed1980

Effect of pyridoxal 5-phosphate on carbidopa and decarboxylation of levodopa.

To study the interaction between enzymes and coenzymes and carbidopa and levodopa, a paired-ion high-pressure liquid chromatographic method was developed to separate carbidopa, dopamine, levodopa, and pyridoxal 5-phosphate. Carbidopa strongly reacted with pyridoxal 5-phosphate, contrary to a previous report. Tyrosine decarboxylase and tyrosine decarboxylase apoenzyme had some activity against levodopa, which could be increased by adding pyridoxal 5-phosphate to the mixture. This activity was inhibited by hydroxylamine hydrochloride.

Apoenzymes↗

PubMed1976

Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli.

An improved purification procedure for the beta2 subunit of tryptophan synthase from from Escherichia coli has led to an essentially pure and stable preparation with a specific enzymatic activity that is 30% higher than the previously reported maximum value. Sedimentation analysis shows that the apo-beta2 subunit is monodisperse and dimeric down to a concentration of 0.02 mg of protein/ml. The binding of pyridoxal 5'-phosphate (pyridoxal-P) to the apo-beta2 subunit and to the alpha2-apo-beta2 complex was studied by equilibrium dialysis and spectroscopic titration. Both the beta2 subunit and the alpha2beta2 complex bind 2 mol of pyridoxal-P with no unspecific binding observable at higher concentrations of pyridoxal-P. The binding of pyridoxal-P to the apo-beta2 subunit is cooperative (Hill coefficient nH = 1.7). The data have been fitted to the Adair equation, yielding the apparent microscopic dissociation constants for the complexes with one and two bound ligand molecules. They differ by a factor of 38, suggesting that the apo- and holo-beta2 subunits have distinct conformations. The binding of pyridoxal-P to the alpha2-apo-beta2 complex is noncooperative with a value of the dissociation constant intermediate between the two values of the beta2 subunit. This finding suggests that the alpha subunit may stabilize a third conformational state of the beta2 subunit.

Apoproteins↗

PubMed1984

Comparison of pyridoxal phosphate-supplemented reagent kits for alanine aminotransferase using centrifugal analysis.

The addition of pyridoxal-5-phosphate for assay of alanine aminotransferase has been recommended. The referral methods are inconvenient for high volume instrumentation due to use of multiple reagents and blanks. We adapted a well-documented method to the centrifugal analyzer as a reference for adaptation of two kit methods. Reference intervals obtained and linearity determined were all similar. The kit methods compared favorably to the referral method. Linear regression analysis yielded the following: DOW = 1.011 BERG + 1.666, r = 0.9965; SKI = 0.938 BERG + 4.559, r = 0.9926; and DSKI = 1.028 BERG + 1.051, r = 0.9889. Precision of the assays was acceptable. We concluded that automation of reagent kits incorporating pyridoxal-5-phosphate is feasible and the assays compared favorably to a recommended method. High volume instrumentation can be used without denigration of analytical quality and to allow comparability to a documented method for interlaboratory review.

Adult↗

PubMedDate not supplied

[Localization of pyridoxal-5-phosphate, covalently bound with human hemoglobin. Spectrofluorimetric studies].

Human apohemoglobin tryptophan residues were localized in the regions of the protein globule with restricted mobility. By the method of dynamic quenching of phosphopyridoxyl chromophore fluorescence, the heterogeneity of pyridoxal-5-phosphate molecules covalently bound to the human hemoglobin molecules was determined from the accessibility to solvent. The first four pyridoxal-5-phosphate molecules are localized in the hydrophobic regions of the hemoglobin molecule; at the same time, they have a high mobility. One of these molecules is situated at the site inaccessible to the solvent, which coincides with the anion-binding center of the oxyhemoglobin molecule. The next pyridoxal-5-phosphate molecules modify the surface amino groups of the protein. In the apohemoglobin molecule, the pyridoxal-5-phosphate binding sites are more exposed to the solvent, as compared to hemoglobin. In the hemoglobin molecule modified by pyridoxal-5-phosphate, an effective electron excitation energy transfer from tryptophan residues to phosphopyridoxyl chromophores occurs. The effective distances between tryptophanyls of single subunits of hemoglobin and the covalently bound pyridoxal-5-phosphate molecule were estimated to be 19 A for the alpha-subunit and 17 A for the beta-subunit.

Hemoglobins↗

PubMed1993

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling.

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

[Mechanism of interaction of pyridoxal-5-phosphate with human serum albumin].

Four and five molecules of pyridoxal-5-phosphate are irreversibly bound to human serum albumin in neutral aqueous or aqueous-spirituous media, respectively. The first step is a reversible formation of the Schiff bases with lysine residues. It is followed by an irreversible interaction of aldimine bond with an imidazole ring of the neighbouring histidine to form cyclic compounds of substituted aldimine type. Cyclic products of the reaction are characterized by absorption spectra with a maximum at 330 nm and fluorescence spectra with a maximum at 400-410 nm in a neutral medium.

Amino Acid Sequence↗

PubMed1986

Plasma pyridoxal 5'-phosphate in women with the premenstrual syndrome.

Measurements of plasma pyridoxal 5'-phosphate (PLP) were made in 210 healthy premenopausal women, and related to their experience of the premenstrual syndrome (PMS). The 41 women (20 per cent) who had moderate or severe PMS were compared with 95 women (45 per cent) with no, or only slight, premenstrual symptoms: the former group were older and were more likely to have children, but there were no significant differences in smoking habits or mode of contraception. Plasma PLP values were similar in the two groups (mean values with s.d. 39.59 +/- 22.95 and 40.56 +/- 23.33 nmol/l respectively). We conclude that pyridoxine status, as measured by plasma PLP levels, is not altered in women with PMS and that pyridoxine deficiency is unlikely to contribute to the occurrence of this syndrome.

Adult↗

PubMed1991

A comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes at a molecular level.

Pyridoxal 5'-phosphate is a coenzyme for a number of enzymes which catalyse reactions at C alpha of amino acid substrates including transaminases, decarboxylases and serine hydroxymethyltransferase. Using the X-ray coordinates for a transaminase, aspartate aminotransferase, and the results of stereochemical and mechanistic studies for decarboxylases and serine hydroxymethyltransferase, an active-site structure for the decarboxylase group is constructed. The structure of the active-site is further refined through active-site pyridoxyllysine peptide sequence comparison and a 3-D catalytic mechanism for the L-alpha-amino acid decarboxylases is proposed. The chemistry of serine hydroxymethyltransferase is re-examined in the light of the proposed decarboxylase mechanism.

Amino Acid Sequence↗

PubMed1993

Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase.

The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.

Affinity Labels↗

PubMed1998

Nonstereospecific transamination catalyzed by pyridoxal phosphate-dependent amino acid racemases of broad substrate specificity.

Pyridoxal 5'-phosphate-dependent amino acid racemases of broad substrate specificity catalyze transamination as a side reaction. We studied the stereospecificities for hydrogen abstraction from C-4' of the bound pyridoxamine 5'-phosphate during transamination from pyridoxamine 5'-phosphate to pyruvate catalyzed by three amino acid racemases of broad substrate specificity. When the enzymes were incubated with (4'S)- or (4'R)-[4'-3H]pyridoxamine 5'-phosphate in the presence of pyruvate, tritium was released into the solvent from both pyridoxamine 5'-phosphates. Thus, these enzymes abstract a hydrogen nonstereospecifically from C-4' of the coenzyme in contrast to the other pyridoxal 5'-phosphate-dependent enzymes so far studied, which catalyze the stereospecific hydrogen removal. Amino acid racemase of broad substrate specificity from Pseudomonas putida produced D- and L-glutamate from alpha-ketoglutarate through the transamination with L-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the nonstereospecific overall transamination between L-ornithine and alpha-ketoglutarate. The cleavage and formation of the C-H bond at C-4' of the coenzyme and C-2 of the substrate thus occurs nonstereospecifically on both sides of the plane of the coenzyme-substrate complex intermediate. Amino acid racemase of broad substrate specificity is the first example of a pyridoxal enzyme catalyzing nonstereospecific transamination.

Amino Acid Isomerases↗

PubMed1976

Differential effects of pyridoxal 5'-phosphate on lysine residues in rabbit and sturgeon muscle aldolases.

Pyridoxal 5'-phosphate (PLP) in aqueous solutions can form a Schiff base complex with 14 and 16 lysine residues of rabbit and sturgeon muscle aldolases (EC 4.1.2.13), respectively. Although the mechanism of their interaction with PLP should be the same, these residues can be differentiated into three families on the basis of their inhibition constant Ki and rate constant k. The lysine residues of one of these families do not react with PLP in the presence of the substrates. Therefore, they are assumed to be part of the active center. In the sturgeon muscle aldolase, 3.7 substrate protected lysine residues are present. Rabbit aldolase, although tetrameric, contains only 2.8 substrate protected lysine residues. This suggests that one active center of this enzyme may be 'buried'. Structural studies showed the following sequence around the substrate protected lysine residues, in the rabbit aldolase: Gly-(Gly2, Val3)-Pyridoxyl Lys-Ile-Asp-Lys.

Amino Acids↗

PubMed1978

Plasma pyridoxal-5'-phosphate levels and clinical correlations in chronic hemodialysis patients.

Plasma pyridoxal-5'-phosphate (PLP) was measured by a specific method in 45 stable, chronic hemodialysis patients and 13 normal adults. Despite oral pyridoxine supplements (1 to 5 mg/day) a majority (64%) of patients had low levels. The difference between normals (8.5 +/- 3.7 ng/ml) and dialysis patients (3.6 +/- 3.6 ng/ml) was significant at P less than 0.01. Plasma PLP appeared to decrease with increasing duration of time on dialysis therapy. In vivo clearance studies as well as pre- and postdialysis plasma levels indicated that PLP was not removed by the dialyzer. Mean plasma PLP levels were normal in patients with stable motor nerve conduction velocity and a low transfusion requirement and low in those with decreasing motor nerve conduction velocity or a high transfusion requirement but the difference between the means in each group was not statistically significant. High oral doses of pyridoxine (100 to 200 mg/day) but not low doses (1 to 5 mg/day) restored PLP levels to normal in a majority of patients after 2 weeks.

Adult↗

PubMed1990

Aging and vitamin B-6 depletion: effects on plasma pyridoxal-5'-phosphate and erythrocyte aspartate aminotransferase activity coefficients in rats.

Plasma pyridoxal-5'-phosphate (PLP) and erythrocyte aspartate aminotransferase activity coefficients were longitudinally determined in rats. Blood was obtained at weeks 0, 1, 2, 4, 6, 9, and 11 from rats initially aged 3 wk, 3 mo, 12 mo, and 23 mo (weeks 0, 1, 4, 6, and 9 only). The diet groups were ad libitum control (ALC), deficient (DEF), and pair-fed control (PF). Plasma PLP concentrations of controls were highest at 3 mo, intermediate at 3 wk and 12 mo, and lowest at 23 mo. When an additional group of 22-mo-old rats was fed a high-vitamin B-6 diet for 4 wk, their low baseline plasma PLP concentrations did not increase significantly. Plasma PLP decreased significantly within 1 wk and activity coefficients increased significantly by week 4 in all DEF rats. Depletion was most rapid and severe in the youngest DEF rats and least in 12-mo-old DEF rats. Mechanisms for the low plasma PLP control values and resistance to depletion in aged rats remain to be determined.

Aging↗

PubMed1984

Mechanism of inactivation of pyridoxal phosphate-linked aspartate transaminase by gostatin.

The inactivation mechanism of pyridoxal phosphate-linked mitochondrial aspartate transaminase (pig heart) by gostatin (5-amino-2-carboxy-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid), a novel amino acid produced by Streptomyces sumanensis, was investigated. Gostatin is a time-dependent inhibitor of the enzyme giving an enzyme half-life of 1.8 min at 3.1 microM (25 degrees C). The kinetic properties of the inhibitor suggest that it is a suicide substrate (mechanism-based inhibitor) of the enzyme, and the observed Ki is 59 microM and Kcat is 0.11S-1 at 25 degrees C. Incubation of the enzyme with a stoichiometric amount of the inhibitor (1 mol of inhibitor/1 mol of enzyme monomer) results in complete inactivation. Spectrophotometric titration and gel filtration experiments indicate the binding of 1 mol of gostatin with 1 mol of enzyme monomer. Gostatin serves as an efficient titrant for the enzyme. Liberation of a compound having inhibitory activity against the apo-form enzyme from the enzyme-inhibitor complex under denaturing conditions suggests irreversible modification of the cofactor.

Amino Acids, Dicarboxylic↗

PubMed1986

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.

Amino Acid Oxidoreductases↗

PubMed1988

Diaminopropionate ammonia-lyase from Salmonella typhimurium. Purification and characterization of the crystalline enzyme, and sequence determination of the pyridoxal 5'-phosphate binding peptide.

We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5'-phosphate(pyridoxal-P)/mol of enzyme. The enzyme catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyridoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme and other pyridoxal-P-dependent enzymes.

Amino Acids↗

PubMed1975

Inhibition of horse muscle acylphosphatase by pyridoxal 5'-phosphate.

It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosphatase at or near the active site of the enzyme.

Amino Acids↗

PubMed1980

Effect of vitamin B6 deficiency on in vitro uptake and metabolism of pyridoxine x HCL by rat jejunum.

The effect of vitamin B6 deficiency on the uptake and metabolism of pyridoxine x HCl by rat jejunum was studied in vitro utilizing everted sacs. Rats were studied in three grops: control, B6-replenished, and B6-deficient. Plasma and jejunal tissue pyridoxal phosphate levels in deficient rats were profoundly depressed compared to control and replenished groups. Uptake and phosphorylation rates for pyridoxine x HCl were similar among the three groups. Jejunal pyridoxal phosphate levels were unchanged after incubation of sacs from control and replenished rats for 30 min in 2 and 20 micro M pyridoxine x HCl under low phosphate (1.1 mM) conditions. Jejunal pyridoxal phosphate significantly increased, however, during similar 30-min incubations in sacs of deficient rats under low phosphate conditions and in control sacs under high phosphate (80 mM) conditions. Conclusions are 1) in vitro uptake and phosphorylatin remain intact during vitamin B6 deficiency; 2) jejunal pyridoxal phosphate is maintained at a constant level in sacs from control and replenished rats in spite of ongoing uptake and phosphorylation of pyridoxine; 3) jejunal pyridoxal phosphate significantly increases during incubationin sacs from vitamin B6-deficient rats; and 4) absorbed pyridoxine can serve directly as the source for this increase.

Animals↗