Inactivation of rat brain acetylcholinesterase by pyridoxal 5′-phosphate

Effects of pyridoxal 5′-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5′-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5′-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5′-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5′-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5′-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5′-phosphate could be reversed upon exhaustive dialysis of the pyridoxan 5′-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5′-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.     

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate

Homogeneous D-ribulose 1,5-bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, $\text{Chlamydomonas reinhardtii}$, and $\text{Hydrogenomonas eutropha}$ are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from $\text{Rhodospirillum rubrum}$, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from $\text{Rhodospirillum rubrum}$.     

Acetylcholinesterase is not inhibited by pyridoxal 5′-phosphate

Acetylcholinesterase activity was assayed in the absence and presence of pyridoxal 5−phosphate. If substrate hydrolysis was measured by the pH-stat method, its rate was not significantly affected by pyridoxal 5′-phosphate. In the spectrophotometric assay, however, this compound led to an apparent decrease in rate. The discrepancy between the two assays is explained by stray-light artefacts produced by pyridoxal 5′-phosphate at the wavelenghts of the spectrophotometric assay.     

Desensitization to glucose 6-phosphate of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal 5′-phosphate

Incubation of the nonphosphorylated form of maize-leaf phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5′-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7±0.3 s⁻¹ M⁻¹ and a first-order reverse rate constant of 0.0046±0.0002 s⁻¹. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.     

Semi-rational chemical modification of endoinulinase by pyridoxal 5′-phosphate and ascorbic acid

It is important to improve the quality of the enzyme inulinase used in industrial applications without allowing the treatment to have any adverse effects on enzyme activity. We achieved preferential chemical modification of the non-catalytic domain of endoinulinase (EC 3.2.1.7) to enhance the thermostability of the enzyme. We used pyridoxal 5′-phosphate (PLP) to modify the more accessible lysine residues at the surface of endoinulinase and then performed a necessary step of reduction with ascorbate. Endoinulinase was incubated in the presence of PLP at various concentrations; this step was followed by reduction of the resulting Schiff base and dialysis. The effects of different PLP concentrations and incubation times on enzyme modification were evaluated. Enzyme deactivation was observed immediately after treatment, even at low PLP concentrations, while reactivation was observed for samples treated with low PLP concentrations after a period of time. Structural analysis revealed that the α-helix content increased from 13.60% to 17.60% after applying the modification strategy; consequently, enzyme stabilization was achieved. The melting temperature (T_m) of the modified enzyme increased from 64.1 °C to 72.2 °C, and a comparative study of thermal stability at 25 °C, 45 °C, and 50 °C for 150 min confirmed that the enzyme was stabilized because of increase in its half-life (t_1/2) after PLP modification/ascorbate reduction. The modification process was optimized to achieve the optimum mole ratio for the PLP/endoinulinase (1.37). Excess moles of the modifier are thought to be responsible for enzyme deactivation through unwanted/nonspecific and noncovalent interactions, and the optimization ensured that there was no excess modifier after the desired covalent reaction was complete.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5′-phosphate

1. The alpha and beta subforms of aspartate aminotransferase were purified from pig heart. 2. The alpha subform contained 2mol of pyridoxal 5'-phosphate. The apo-(alpha subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(alpha subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The beta subform had approximately half the specific enzyme activity of the alpha subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(beta subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.     

Essential primary amino groups of ribulose bisphosphate carboxylase/oxygenase indicated by reaction with pyridoxal 5′-phosphate

Pyridoxal 5′-phosphate (PLP) and CO₂ were competitive for the same site on d-ribulose bisphosphate carboxylase (EC 4.1.1.39), presumably an ?-amino group of a lysyl residue. An apparent noncompetitive inhibition occurred with respect to ribulose bisphosphate (RuP₂). The time course of inactivation was first order with respect to both PLP and RuP₂ carboxylase concentration. The extinction coefficient was found to be 5800 ± 800 m^?1 cm^?1 for the maximal absorbance at 432 nm of the enzyme/PLP Schiff base. The titration of the enzyme with PLP gave a biphasic double reciprocal plot. The number of amino groups reacting with PLP was calculated to be 9.5 and 19 per molecule of enzyme, at low and high concentrations of PLP. Half of the amino groups available for reaction with PLP at either concentration could be protected by RuP₂. When the RuP₂ carboxylase/PLP complex was reduced with NaBH₄ in the absence of substrates, only 20% of the enzyme activity was recovered, but, in the presence of RuP₂ or bicarbonate, the recovery of enzyme activity was 80 or 25%, respectively. It is concluded that there are eight primary groups that react with PLP, one at each of the eight catalytic sites of the RuP₂ carboxylase molecule. It is postulated that this amine is essential for formation and/or activation of an enzyme/CO₂ complex. In the absence of CO₂, this amine may react with the carbonyl function of RuP₂. This provides an explanation for the inactivation of RuP₂ carboxylase upon preincubation with RuP₂ in the absence of CO₂ and even the inactivation of the activated enzyme at RuP₂ concentrations above 0.5 mm. The second set of eight primary amino groups which react with PLP may involve the CO₂ regulatory site.     

Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5′-phosphate: evidence for an allosteric modulation of neuraminidase activity

Incubation of Sendai virus with pyridoxal 5′-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of hemagglutinin-neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663–668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.     

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.     

The control of plant glutamate dehydrogenase by pyridoxal-5′-phosphate

The proposition that the nitrogen status of a plant is reflected by the ratio pyridoxal phosphate to pyridoxamine phosphate and that this ratio exerts a controlling influence on plant metabolism has been examined. The ratio pyridoxal phosphate to pyridoxamine phosphate has been shown to increase during nitrogen starvation. The inhibition of glutamate dehydrogenase by pyridoxal phosphate has been examined and the kinetics of inhibition are discussed in relation to the proposed control of metabolism.     

SAR of non-hydrolysable analogs of pyridoxal 5′-phosphate against low molecular weight protein tyrosine phosphatase isoforms

Kinases and phosphatases are key enzymes in cell signal transduction pathways. Imbalances in these enzymes have been linked to numerous disease states ranging from cancer to diabetes to autoimmune disorders. The two isoforms (IFA and IFB) of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) appear to play a role in these diseases. Pyridoxal 5′-phosphate (PLP) has been shown to act as a potent but, impractical micromolar inhibitor for both isoforms. In this study, a series of non-hydrolysable phosphonate analogs of PLP were designed, synthesized and tested against the two isoforms of LMW-PTP. Assay results demonstrated that the best inhibitor for both isoforms was compound 5 with a K_is of 1.84 μM (IFA) and 15.6 μM (IFB). The most selective inhibitor was compound 16, with a selectivity of roughly 370-fold for IFA over IFB.     

Spectrophotometric determination of pyridoxal and pyridoxal 5′-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay

A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.     

Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine

Summary The accessibility of pyridoxal 5′-phosphates of the phosphorylaseab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of theb anda forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylasea andab to resolution at 0°C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of theab hybrid was similar to that of thea and differed from that of theb forms in that it occurred at 30°C and 37°C but not at 0°C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of theab form between that of theb anda forms. We conclude that partial phosphorylation of phosphorylaseb elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5′-phosphates. Resistance ofab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylaseb anda.     

The reactions of pyridoxal 5′-phosphate with the M4 and H4 isoenzymes of pig lactate dehydrogenase

The H₄ and M₄ isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5′-phosphate. In the early stages, inactivation is largely reversible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5′-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Neither pyruvate nor lactate alone gives any protection. With the M₄ isoenzyme, complete protection against inactivation by pyridoxal 5′-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H₄ isoenzyme. In the course of irreversible inactivation of H₄ lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to resolve earlier controversy.     

Chemoenzymatic synthesis of 1-deaza-pyridoxal 5′-phosphate

The first synthesis of 1-deaza-pyridoxal 5′-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate) is described. The chemoenzymatic approach described here is a reliable route to this important isosteric pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyridoxal 5′-phosphate dependent enzymes.     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

Template-directed oligomerization of 3-isoadenosine 5′-phosphate

Summary Template-directed oligomerization of an activated derivative of 3-isoadenosine 5-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3–5-linked oligomers while the reaction of ImpA gives only 2–5-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.