Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

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.     

Structural role of pyridoxal 5'-phosphate, pyridoxal 5'-phosphate analogs, and other agents in the association of subunits of Bacillus alvei apotryptophanase.

Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.     

A novel coenzymatic function of pyridoxal 5'-phosphate

Pyridoxal 5′-phosphate, the vitamin B₆ derivative, acts as the coenzyme of many enzymes involved in amino acid metabolism. Exceptionally, this compound was found covalently bound to glycogen phosphorylase, the key enzyme in the regulation of glycogen metabolism. Although it is essential for the function of phosphorylase, its direct role has remained an enigma. We have recently found that the glucose moiety of pyridoxal (5′)diphospho (1)-α-D -glucose, a conjugate of pyridoxal 5′-phosphate and glucose 1-phosphate through a pyrophosphate linkage, is transferred to the nonreducing end of glycogen, forming a new α-1,4-glucosidic linkage. This finding emphasizes the importance of the direct phosphate-phosphate interaction between the coenzyme and the substrate in the phosphorylase catalytic reaction. We have proposed a catalytic mechanism for phosphorylase in which the phosphate group of pyridoxal 5′-phosphate acts as an electrophile to the phosphate group of glucose 1-phosphate. This appears to represent the first instance of the direct involvement of a phosphate group in catalysis by enzymes.     

Design of pyridoxal 5'-phosphate dependent catalytic antibodies

Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5"-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme–substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme–substrate compounds and corresponding apoenzyme complexes are reviewed.     

Regulation of pyridoxal 5'-phosphate metabolism in liver

The pyridoxal 5′-phosphate content of liver $\text{in vivo}$ and of hepatocytes $\text{in vitro}$ remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.     

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

Enzymatic dtermination of pyridoxal 5'-phosphate with gamma-cyanoaminobutyric acid aposynthase.

A rapid alternative method is presented for the determination of pyridoxal 5'-phosphate (pyridoxal-P). The method involves the colorimetric analysis of thiocyanate liberated from S-cyanohomocysteine (Hcy (CN)) in the presence of cyanide when catalyzed by the pyridoxal-P dependent enzyme, gamma-cyano-alpha-aminobutyric acid (gamma-CNabu)-synthase (Hcy (CN) thiocyano-lyase [adding CN]). The rate of formation of thiocyanate is determined by the increase in absorbance at 470 nm on treatment of the enzymatic reaction mixture with FeCl3.     

A bacterial phenylalanine aminotransferase lacking pyridoxal 5'-phosphate as cofactor.

A bacterium has been isolated from soil which metabolises phenylalanine initially through the action of a phenylalanine aminotransferase. This enzyme has been purified by conventional techniques and affinity chromatography and shown to be unusual among aminotransferases in not containing pyridoxal 5'-phosphate as cofactor.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

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 wavelengths of the spectrophotometric assay.