Covalent modification of the solubilized rat liver vitamin K-dependent carboxylase with pyridoxal-5′-phosphate

The carboxylation of the pentapeptide substrate, Phe-Leu-Glu-Glu-Ile, by a rat microsomal vitamin K-dependent carboxylase was stimulated two- to threefold at pyridoxal-5′-P concentrations between 0.5 and 1.0 mm. This stimulation was reduced at concentrations higher than 1.0 mm. The K_m for the pentapeptide was lowered twofold in the presence of 1 mm pyridoxal-5′-P. The activation by pyridoxal-5′-P is specific, as 1 mm pyridoxal, pyridoxine, pyridoxine-5′-P, pyridoxamine, pyridoxamine-5′-P, or 4-pyridoxic acid did not stimulate the pentapeptide carboxylation rate. All six analogs, as well as formaldehyde and acetaldehyde, inhibited the carboxylation reaction in a concentration-dependent manner. The activation of the carboxylase by pyridoxal-5′-P appeared to be mediated by its direct binding to the enzyme via Schiff base formation. Sodium borohydride reduction of solubilized microsomes in the presence of pyridoxal-5′-P, followed by dialysis to remove unbound material, resulted in a carboxylase preparation with a specific activity twice that of the untreated control microsomes. The derivatized enzyme was not further stimulated by added pyridoxal-5′-P. This derivatized carboxylase could be obtained in the absence of pentapeptide and divalent cations. The stimulation of the carboxylase activity by divalent cations and pyridoxal-5′-P was mediated at separate site(s) on the enzyme. Studies of the NH₂-terminal pyridoxalated pentapeptide with both a normal and PLP-modified enzyme, in the presence and absence of PLP, demonstrated competition of the pentapeptide PLP moiety to a PLP site on the enzyme. It was concluded that pyridoxal-5′-P forms a covalent attachment to an ?-NH₂ of a lysine near the active site of the carboxylase.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Evidence for essential primary amino groups in a bacterial coupling factor F1ATPase

We have found that the binding of pyridoxal-5′-phosphate to 6 primary amino groups leads to the inactivation of the enzyme. A preferential reaction of pyridoxal-5′-phosphate with the α-subunits of this enzyme can be demonstrated. The reactivity of the amino groups is influenced by various effectors. In the presence of ATP the inhibition of the ATPase activity is noncompetitive.     

Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH₄. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH₄. One mole of N-?-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.     

An affinity labeling reagent for pyridoxal phosphate dependent enzymes.

An analogue of pyridoxal-5′-phosphate, 4′-N-(2,4 dinitro-5-fluorophenyl) pyridoxamine-5′-phosphate, has been synthesised and has been shown to behave as an affinity labeling reagent for the apoenzymes of aspartate and tyrosine aminotransferases, tyrosine decarboxylase and tryptophanase. Of the enzymes tested only apocystathionase is not irreversibly inhibited by the reagent.     

Inhibition by pyridoxal-5'-phosphate of gamma-aminobutyric acid receptor binding to synaptic membranes of cat cerebellum.

The binding of $\text{[}{}^3\text{H]γ-aminobutyric}$ acid to cat cerebellar membranes is $\text{reversibly}$ inhibited in a competitive manner by pyridoxal-5′-phosphate present during the binding assay. Structural analogues of the inhibitor have no such effect. If, on the other hand, the membranes are preincubated with pyridoxal-5′-phosphate followed by the addition of sodium borohydride, a rapid, $\text{irreversible}$ inhibition of subsequent γ-aminobutyric acid binding is observed. Since pyridoxal-5′-phosphate is known to inactivate certain enzymes by reacting with essential lysine residues, the present results suggest that such a lysine residue may be present within the γ-aminobutyric acid receptor.     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

An immunochemical analysis of the dissociation of the beta2 component of Escherichia coli tryptophan synthetase

Conversion of E. coli tryptophan synthetase β₂ subunit to an enzymatically inert apo-derivative is accompanied by a change in immunochemical reactivity. Restoration of the cofactor, pyridoxal-5′-phosphate, simultaneously restores the enzymatic and serological characteristics to normal. This change in antigenic structure is detectable by micro-complement fixation but not by macro-complement fixation or precipitin analysis. Evidence was obtained that: (1) with respect to the antisera used in the experiments, pyridoxal-5′-phosphate is neither an antigenic determinant nor part of a determinant; (2) the reversible shift in antigenic structure can best be explained by the enhanced dissociation of the apoenzyme to a strongly cross-reacting monomer; and (3) the tertiary structure of the monomer remains essentially unchanged upon dissociation.     

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.     

Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI)

Abstract

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E?150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5′-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5′-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range.     

Inactivation and degradation of yeast glucose-6-phosphate dehydrogenase selectively modified by pyridoxal-5-phosphate

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.     

Interaction of pyridoxal-5-phosphate with human serum albumin and pancreatic ribonuclease

Pyridoxal-5-phosphate (in a lesser degree, pyridoxal) interacts with both non-protonated and protonated exposed epsilon-amino groups of lysine residues and with alpha-amino groups in human serum albumin and pancreatic ribonuclease A. The reaction of Schiff base formation proceeds within a wide pH range--from 3.0 to 12.0. At a great pyridoxal-5-phosphate excess in ribonuclease A in neutral or slightly acidic aqueous media all the ten epsilon-amino groups of lysine residues and the alpha-amino groups of Lys-1 become modified. The formation of aldimine bonds of pyridoxal-5-phosphate with protonated amino groups in acidic media is determined by ionization of its phenol hydroxyl and phosphate residues. Acetaldehyde, propionic aldehyde and pyridine aldehyde interact only with non-protonated amino groups of the proteins. The equilibrium constants of pyridoxal-5-phosphate and other aldehydes binding to proteins and amino acids were determined. The rate constants of Schiff base formation for pyridoxal-5-phosphates with some amino acids and primary sites of proteins for direct and reverse reactions were calculated.     

Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase

The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-Leu-Glu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnCl2 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation's ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.     

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5′-phosphate-containing enzyme, but the requirement of pyridoxal-5′-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5′-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5′-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5′-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5′-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5′-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5′ phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5′-phosphate that is present in the mammalian and bacterial enzymes.     

Pyrroline-5-carboxylate reductase from Cucurbita cotyledons

Pyrroline-5-carboxylate reductase, which required reduced pyridine nucleotide and Δ′-pyrroline-5-carboxylate for proline synthesis, was isolated from pumpkin cotyledons. The enzyme was found in the soluble fraction and had a 4.5-fold greater activity with NADH than NADPH. The enzyme was inhibited by NH₂OH, NADP, ATP and slightly by proline. Glutathione or pyridoxal-5-phosphate had little effect on enzyme activity. The enzyme had a pH optimum between 7·0 and 7·6 and was not inhibited by high concentrations of NADH or Δ′-pyrroline-5-carboxylate.     

Evidence for the involvement of a rapidly turning over protease in the degradation of cytochrome oxidase in Neurospora crassa

The reaction of L-aromatic aminoacid decarboxylase (EC 4.1.1.28) with α-methyl-L-DOPA or 5-hydroxy-L-tryptophan leads to the formation of dihydroxyphenylacetone or, respectively, 5-hydroxyindolacetaldeyde. These are produced in amounts far exceeding, on molar basis, that of the coenzyme, pyridoxal-5′-phosphate. The reaction cannot therefore be simply a decarboxylation-dependent transamination, using the coenzyme as an amino group acceptor. Evidence is presented which rules out the possibility that this phenomenon is due to an oxidative deamination.     

Spin label studies on phosphorylase B.

Spin label studies upon phosphorylase B have revealed that the temperature-dependent equilibria between different conformational states are strongly influenced by the presence of substrate or allosteric modifier. In the absence of substrate or modifier, the equilibrium involves only two states, while at least three are involved in their presence. Removal of pyridoxal-5′-phosphate produces a conformational change reflected by increased mobility of the label. The apo-enzyme undergoes a structural change in the presence of the allosteric modifier AMP.     

Effect of vitamin preparations on epileptic activity

It has been shown in experiments on mice and rats that daily administration of subthreshold doses of pentylenetetrazol led to a progressive increase in the sensitivity to the action of the epileptogen, augmentation of brain epileptization and development of the pharmacological kindling. Single administration of nicotinamide in doses of 250, 500 and 1000 mg/kg and alpha-tocopherol in a dose of 100 mg/kg exerted a pronounced antiepileptic effect under the conditions of over kindling. On combined use of nicotinamide and pyridoxal-5-phosphate, nicotinamide, pyridoxal-5-phosphate and alpha-tocopherol the antiepileptic action was more demonstrable. Daily administration of a complex of the drugs (nicotinamide, pyridoxal-5-phosphate, alpha-tocopherol) produced a substantial reduction in epileptic activity under the conditions of overt kindling. The possibility has been demonstrated of preventing the development of epileptic activity with nicotinamide under kindling.     

STOFFWECHSELUNTERSUCHUNGEN DES CEREBRALEN ANFALLSGESCHEHENS

Abstract— The activity of glutamate decarboxylase in the brain of rats during and prior to experimentally produced cerebral seizures was compared with that of control rats. An inhibition of enzyme activity during the tonic convulsions after intracisternal injection of l -glutamate or pyridoxal-5-phosphate, after audiogenic stimulation, after intraperitoneal injection of pentamethylenetetrazole and during the electroshock could be observed. During the preconvulsive stage the enzyme was strongly inhibited after an intracisternal injection of l -glutamate, l -aspartate, and after audiogenic stimulation. Only after the intracisternal injection of pyridoxal-5-phosphate the enzyme activity as compared with that of control rats was unchanged. The different effects of l -glutamate and pyridoxal-5-phosphate in vivo and in vitro on the glutamate decarboxylase are pointed out in particular. The inhibition of this enzyme in vivo is believed to be one of the possible causes of cerebral seizures.     

Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH₄. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH₄ resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB³H₄ resulted in incorporation of ³H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R_f identical to that of authentic N⁶-pyridoxyllysine. When [4-³H]pyridoxal phosphate was used all of the ³H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ?-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4-³H]pyridoxal-5′-phosphate/NaBH₄ and by pyridoxal-5′-phosphate/NaB³H₄. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.