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PubMed1989

Inhibition of thymidylate synthase by pyridoxal phosphate.

1. Pyridoxal phosphate (PLP) reversibly inhibited thymidylate synthase from Lactobacillus casei with a KI of 0.6-0.9 microM. 2. The inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates. 3. The spectrum of PLP was altered by the addition of thymidylate synthase. The spectral changes suggest formation of a thiohemiacetal with an enzyme sulfhydryl group rather than Schiff base formation with a lysine side chain.

Binding, Competitive↗
PubMed2005

Reaction specificity in pyridoxal phosphate enzymes.

Pyridoxal phosphate enzymes catalyze a wide variety of reaction types on amines and amino acids, generally by stabilizing carbanionic intermediates. This makes them very useful in cellular metabolism, but it also creates problems in controlling the reaction pathway that a given enzyme follows, i.e., in controlling reaction specificity. Stereoelectronic effects have been proposed to play a major role in determining the bond to Calpha that gets broken in the external aldimine intermediate that is common to all PLP enzymes. Here, we discuss our work on dialkylglycine decarboxylase aimed at providing direct evidence for stereoelectronic control of external aldimine reactivity. Once a bond to Calpha has been broken to form the carbanionic intermediate, enzymes must also carefully control the fate of this reactive species. Our studies with alanine racemase suggest that the enzyme selectively destabilizes the carbanionic quinonoid intermediate to promote higher racemization specificity by avoiding transamination side reactions.

Alanine Racemase↗
PubMed1974

Vitamin B6 metabolism in chronic alcohol abuse. Pyridoxal phosphate levels in plasma and the effects of acetaldehyde on pyridoxal phosphate synthesis and degradation in human erythrocytes.

The plasma pyridoxal-5'-phosphate (PLP) level of alcoholic subjects has been compared with that of non-alcoholic individuals in order to ascertain the incidence of abnormal vitamin B(6) metabolism in chronic alcohol abuse. 66 alcoholic subjects were selected on the basis that they did not exhibit abnormal liver function tests and hematologic findings. 35 of them had plasma PLP concentrations less than 5 ng/ml, the lowest value encountered in 94 control subjects, indicating a high incidence of deranged PLP metabolism in alcoholic patients even when hepatic and hematologic abnormalities are absent. The biochemical basis for the altered PLP metabolism in chronic alcohol abuse was examined. Low plasma PLP levels in alcoholics were not accompanied by decreased pyridoxal kinase and pyridoxine phosphate oxidase activities in erythrocytes. Further studies with erythrocytes demonstrated that the cellular content of PLP is determined not only by the activities of these PLP-synthesizing enzymes but also by the activity of a phosphate-sensitive, membrane-associated, neutral phosphatase, which hydrolyzes phosphorylated B(6) compounds.Acetaldehyde, but not ethanol, impaired the net formation of PLP from pyridoxal, pyridoxine, and pyridoxine phosphate by erythrocytes. However, when the B(6)-phosphate phosphatase activity was inhibited by 80 mM phosphate, this effect of acetaldehyde was abolished. By the use of broken cell preparations, it was possible to demonstrate directly that the action of acetaldehyde is mediated by the phosphatase, resulting in an acceleration of the degradation of the phosphorylated B(6) compounds in erythrocytes.

Acetaldehyde↗
PubMed1976

Pyridoxal phosphate. 5. 2-Formylethynylphosphonic acid and 2-formylethylphosphonic acid, potent inhibitors of pyridoxal phosphate binding and probes of enzyme topography.

The title compounds (17 and 21) were prepared in good yield by synthesis of the phosphonate diester acetals (14 and 19), deesterification with chloro- or bromotrimethylsilane, hydrolysis of the acetal group, and formation of the characterized barium salts. The 3-carbon aldehydrophosphonic acids (17 and 21) were potent inhibitors (Ki = 2 X 10(-6)) of pyridoxal phosphate (PPal) binding to apoaspartate aminotransferase (AAT) and are believed to span between and bind to the enzymic functional groups which bind the aldehyde and phosphate groups of PPal.

Apoenzymes↗
PubMed1978

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.

Adenosine Diphosphate Glucose↗
PubMed1971

Photoinactivation of aldolases by pyridoxal phosphate and its analogues.

Pyridoxal phosphate can act as a specific photosensitizer for amino acid residues in rabbit muscle and spinach leaf aldolases, but the residues affected depend on the pH of the reaction. Below pH 8 one histidine residue per enzyme subunit is destroyed; above pH 8.5 there is little loss of histidine, and photoinactivation is associated with the destruction of specific tyrosine residues, particularly the COOH-terminal residues. Pyridoxal and 4-pyridinecarboxaldehyde are much less effective than pyridoxal phosphate at neutral pH, but are similar to pyridoxal phosphate in their photosensitizing activity at the higher pH. Compounds lacking the aldehyde group or the pyridine ring show little or no activity. A number of other enzymes, including alpha-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and yeast hexokinase, were also photoinactivated in the presence of pyridoxal phosphate; however, rabbit liver aldolase and two isomerases tested were completely resistant. The results suggest that certain enzymes, including rabbit muscle and spinach aldolases, but not rabbit liver aldolase, contain a specific site which interacts with pyridoxal phosphate, and that the conformation of this site changes in the pH range between 8.0 and 8.5

Amino Acids↗
PubMed1975

Topology of binding sites for carbamyl phosphate in aspartate transcarbamylase from Escherichia coli. The use of pyridoxal phosphate as covalent probe.

Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.

Aspartate Carbamoyltransferase↗
PubMed1986

Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal.

Pyridoxal phosphate reacts with not only the lysyl residue(s) essential for enzymatic activity but also other reactive lysyl residues in rabbit muscle lactate dehydrogenase (EC 1.1.1.27). To raise the specificity of pyridoxal phosphate, adenosine diphospho-, triphospho-, and tetraphosphopyridoxals have been newly synthesized and used for modification of the enzyme. Incubation of the enzyme for 30 min with the diphospho, triphospho, and tetraphospho compounds all at 1 mM followed by reduction by sodium borohydride resulted in the loss of enzymatic activity by 64, 51, and 34%, respectively. NADH almost completely protected the enzyme from inactivation, whereas pyruvate showed no protection. Binding of the reagents to the enzyme subunit in an equimolar amount corresponds to the complete inactivation. The adenosine diphosphopyridoxal modified enzymes with different residual activities were chromatographed on a Blue Toyopearl affinity column. The results showed the presence of at least four enzyme species besides the intact enzyme that are significantly different from one another in the amount of the reagent bound, the affinity for NADH, and the specific activity. The decrease in the affinity of the enzyme for NADH and the loss of enzymatic activity paralleled in the modification by adenosine diphosphopyridoxal, whereas, in the modification by pyridoxal phosphate, the decrease in the affinity for NADH preceded the inactivation. It is concluded that modification by adenosine polyphosphopyridoxal compounds are specific for the active site lysyl residue(s) in lactate dehydrogenase.

Adenine Nucleotides↗
PubMed1983

Pyridoxal phosphate as an antisickling agent in vitro.

Although pyridoxal phosphate is known to inhibit gelation of purified hemoglobin S, antisickling activity has never been demonstrated for intact erythrocytes. We incubated washed erythrocytes at 37 degrees C either in buffer alone, or with added pyridoxal phosphate or pyridoxal, washed these cells, suspended them in untreated buffer, and compared the percent modified hemoglobin, the oxygen affinity, and the extent of sickling under hypoxia. Pyridoxal phosphate modified intracellular hemoglobin more slowly than pyridoxal. Pyridoxal phosphate lowered the oxygen affinity of normal cells, but had no effect on oxygen binding by sickle cells. Pyridoxal increased the oxygen affinity of normal and sickle erythrocytes equally. Pyridoxal phosphate significantly inhibited sickling of sickle or sickle trait erythrocytes (P less than 0.001). Inhibition of sickling by pyridoxal phosphate was largely independent of oxygen binding; whereas inhibition of sickling by pyridoxal was almost entirely dependent on increased oxygen binding. Although pyridoxal phosphate and pyridoxal both inhibit sickling by modification of hemoglobin S, they differ in the kinetics of whole cell modification, the effect on oxygen affinity of intact cells, and the mechanism of action of the antisickling activity.

Anemia, Sickle Cell↗
PubMed1990

Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue.

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.

Adenosine Diphosphate↗
PubMed2005

Pyridoxal phosphate inhibits the group I intron splicing.

The coenzyme pyridoxal phosphate and its analogs were tested for inhibition of the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td). Of all compounds examined, the pyridoxal phosphate was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: pyridoxal phosphate > pyridoxal > pyridoxine > pyridoxamine > pyridoxic acid. Increasing Mg2+ concentration up to 14 mM overcame the suppression of self-splicing by pyridoxal phosphate up to 95% of the level of normal splicing, implying its interference with effective catalysis of Mg2+. The kinetic analysis demonstrated that pyridoxal phosphate acts as a mixed type noncompetitive inhibitor for the td intron RNA with a K(i) of 11.8 mM. The specificity of the splicing inhibition by pyridoxal phosphate is predominantly due to increases in K(m) and decreases in V(max) values.

Bacteriophage T4↗
PubMed1986

Studies on the succinate dehydrogenating system. Interaction of the mitochondrial succinate-ubiquinone reductase with pyridoxal phosphate.

The inhibitory effect of pyridoxal phosphate on the Triton X-100 solubilized purified bovine heart succinate-ubiquinone reductase (Choudhry, Z.M., Gavrikova, E.V., Kotlyar, A.B., Tushurashvilli, P.R. and Vinogradov, A.D. (1985) FEBS Lett. 182, 171-175) was studied. The kinetics of the enzyme inactivation by pyridoxal phosphate was found to be strongly dependent both qualitatively and quantitatively on the concentration of the protein-detergent complexes. In the diluted system the inactivation of the ubiquinone-depleted enzyme was completely prevented by the saturating concentrations of Q2, carboxin, thenoiltrifluoroacetone and pentachlorophenol, i.e., by the substrate and specific inhibitors of the enzyme. The protective effects of Q2 and the inhibitors was employed to quantitate the affinities of the ligands to their specific binding sites. Strong difference in the affinity of Q2 to the reduced and oxidized enzyme was found. When the soluble reconstitutively active succinate dehydrogenase was treated with pyridoxal phosphate, the reactivity of the enzyme towards low ferricyanide concentrations and its reconstitutive activity was significantly protected against aerobic inactivation.

Animals↗
PubMed2001

The role of pyridoxal phosphate in the function of EspB, a protein secreted by enteropathogenic Escherichia coli.

The sequence of EspB, a secreted protein required for virulence of enteropathogenic Escherichia coli (EPEC), reveals a motif common to enzymes that bind pyridoxal phosphate. Pyridoxal phosphate was not found by fluorometry in concentrated supernatants of EPEC cultures that contain EspB. Plasmids containing cloned espB, in which the lysine residue conserved in the motif was substituted with either an arginine or methionine residue, remained capable of complementing an espB deletion mutant to restore EspB function. The results of these studies do not support a role for pyridoxal phosphate in EspB function.

Actins↗