Rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase. Purification and properties.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been purified 2000-fold from rabbit liver. The enzyme preparation migrates as a single protein and activity band on analytical disc gels containing 4,7, or 9 percent acrylamide, and as a single protein band on sodium dodecyl sulfate acrylamide gels. The oxidase is, therefore, homogeneous by these criteria. The pure enzyme catalyzes the following reactions in the presence of FMN: (See journal for formula). These activities copurify in the ratio of 1:1:1. Apparent K-m values are 10 muM for pyridoxamine-P, 30 muM for pyridoxine-P, and 40 nM for FMN. Apparent K-m values for N-(phosphopyridoxyl)amines range from 3.1 times 10-5 M to 1.6 times 10-3 M. The dissociation constant for FMN binding, determined by quenching of protein fluorescence, is 20 nM. The pH optima for all three types of substrates are broad, with maxima near pH 9. The pH dependence of FMN binding, measured by quenching of flavin fluorescence, has the same shape as the substrate activity profile. The holoenzyme has absorption maxima red-shifted from those of FMN to 380 nm and 448 nm, and exhibits spectral changes typical of flavoproteins upon reduction with dithionite. Its oxidation-reduction potential at pH 7 in phosphate buffer is -0.131 volt. The native enzyme has a molecular weight of 54,000 and is made up of two possibly identical polypeptide chains with molecular weights of 27,000. The applicability of proposed mechanisms of flavin catalysis to this flavoprotein is discussed.     

Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase

Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of approximately 0.2 sec(-1) and K(m) values in the low micromolar range for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. Pyridoxal 5'-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5'-phosphate tightly on each subunit.     

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.     

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H⁺?(dUMP-TSase-H⁺). [1] The enthalpy, ΔH₁′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH₁′ = ?28 kJ mol^?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG₁′ = ?30 kJ mol^?1 (K₁ = 1.7 × 10⁵ m^?1). From these parameters, ΔS₁′ was calculated to be +5 J mol^?1 degree^?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG₁′ = ?35 kJ mol^?1 (K₁ = 1.2 × 10⁶ m^?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n_HH⁺?FdUMP₂ ? TSase ? (H⁺)_nH. [2] The enthalpy for this reaction, ΔH₂, was also measured calorimetrically and found to be ?30 kJ mol^?1 with n_H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.     

Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate.

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.