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.     

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.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

Determination of plasma pyridoxal 5'-phosphate by an enzymatic-high-performance liquid chromatographic procedure

An enzymatic-HPLC procedure for the determination of plasma pyridoxal 5'-phosphate (PLP) has been established. The assay is based on the decarboxylation of L-3,4-dihydroxyphenylalanine using Streptococcus tyrosine decarboxylase apoenzyme, which requires PLP as cofactor. The product of the enzyme reaction, dopamine, is measured by Coulochem electrochemical detection with a series of oxidizing and then reducing electrodes. Trace amounts of PLP in the apoenzyme preparation were removed with the aid of cysteine-sulfinic acid and gel filtration. The detection limit for PLP by this method is 50 pM in plasma.     

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase

In the course of a half-reaction of enzymic transamination, the aldimine adduct formed between the coenzyme pyridoxal 5'-phosphate and the amino acid substrate tautomerizes to the ketimine intermediate which is then hydrolyzed to the oxo acid product and the pyridoxamine 5'-phosphate form of the enzyme. In the reverse half-reaction the tautomerization is initiated by the removal of a proton from the pro-S position at C-4' of the PMP moiety of the ketimine intermediate. The present study investigates the question whether the pro-S hydrogen at C-4' of PMP is labilized by its active site environment independently of the formation of the ketimine intermediate, i.e. in the absence of substrate. Reconstitution of apoaspartate aminotransferase (mitochondrial isoenzyme from chicken) with [4'-3H] PMP results indeed in a stereospecific exchange of pro-S 3H with solvent water. The exchange follows first order kinetics (t 1/2 = 23 min at pH 7.5 and 25 degrees C). Unbound PMP showed no measurable exchange. Rigorous control experiments excluded the possibility that the observed exchange was due to a transamination reaction of the enzyme with contaminating oxo acid substrates. The newly observed stereospecific exchange reaction allows to investigate the acid/base properties of C-4' and the modulating effects of its active site environment independently of the preceding and following steps of enzymic transamination.     

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors

Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.     

N-(5'-phospho-4'-pyridoxyl)amines as substrates for pyridoxine (pyridoxamine) 5'-phosphate oxidase

A preparation of pyridoxine (pyridoxamine) 5′-phosphate oxidase, with a specific activity of 9,400 nmoles/hr/mg protein, 10-fold higher than that previously reported, was used to study the oxidation of various N-(5′-phospho-4′-pyridoxyl)amines. Values for K_m, from 3.1 × 10^?5 M to 1.6 × 10^?3 M, and for V_max, relative to pyridoxamine-P, of 20 to 140% were obtained. Compounds lacking a 5′-phosphate were not substrates, and the enzymic reaction was dependent on the presence of both FMN and O₂. N-(phosphopyridoxyl)-L-amino acids had lower K_m's than the corresponding -D-amino acid compounds. When 1-¹⁴C-N-(phosphopyridoxyl)glycine was used as a substrate, no ¹⁴CO₂ was evolved, and 1-¹⁴C-glycine was detected in the incubation mixture.     

Activation and inactivation of rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase activity by urea and other solutes.

Urea, guanidine hydrochloride, and neutral salts both activate and denature pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) from rabbit liver. Activation occurs at lower concentrations (e.g. 2-2.5 m for urea) of these compounds and is rapid and reversible. Greater structural changes leading to inactivation occur slowly under “activating conditions” but rapidly at higher concentrations of urea. Both reversibly and irreversibly inactivated species are formed. Activation by urea does not involve either dissociation of the enzyme to subunits or aggregation to multimers, and there is little disruption of protein secondary structure. The V and K_m for substrates, K_i for product, and the rate of release of product from the enzyme are increased by urea, and substrate inhibition is decreased; urea has little effect on the reactivity of reduced enzyme with oxygen. Both flavin and tryptophanyl fluorescence increase in the presence of urea; at lower concentrations of urea (≤2 m), there is a rapid increase followed by slower, sigmoidal increases. The polarization of flavin fluorescence of the oxidase is increased upon the addition of 2 m urea, which corresponds to the initial enhancement of protein and flavin fluorescence intensities, and then decreases. The near-ultraviolet-visible absorption spectra of native enzyme and that treated with 2 m urea are only slightly different; however, a considerable change at the flavin-binding site is reflected by the circular dichroism spectra. Hence, it appears that urea yields a rapidly formed, “activated” species of the oxidase that is changed primarily at the active site in a manner that allows increased dissociation of substrate and product.     

Determination of trans-arachidonic acid isomers in human blood plasma

Endogenous trans fatty acids originate from diet, but recent studies also suggest that cis-trans isomerization of fatty acids is possible by nitrogen dioxide radical, a product of NO and nitrite oxidation. We developed a method for quantitative analysis of four trans-arachidonic acids (TAA) in human plasma using isotopic dilution gas chromatography/mass spectrometry (GC/MS) with deuterium-labeled internal standard. Esterification of the plasma fatty acid extract with pentafluorobenzyl (PFB) bromide followed by high-performance liquid chromatography purification yielded a fairly pure fraction containing TAA-PFB esters that was analyzed by GC/MS. Partial separation of the TAA isomers was obtained on various GC columns. Comparison of the retention time with the synthetic standards revealed that all four TAA isomers are present in human plasma. The mean concentration of TAA in human plasma was 20.2ng/ml. The levels of isomers were 12.48+/-1.28, 2.75+/-0.39, and 4.99+/-0.74ng/ml for 5E-AA + 11E-AA, 8E-AA, and 14E-AA, respectively. The identification of TAA in plasma suggests that isomerization of arachidonic acid occurs in vivo. Our method allows distinguishing between the dietary and the NO(2)-dependent mechanisms of trans fatty acid formation and will be useful in defining the role of TAA as an in vivo marker of nitrooxidative stress in clinical and experimental settings.     

Chemical modification of human placental glutathione transferase by pyridoxal 5'-phosphate.

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.     

Evidence for an essential histidyl residue at the active site of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

Pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) from rabbit liver is inactivated by diethylpyrocarbonate in an all-or-none fashion with first order kinetics with respect to modifier concentration. The rate of inactivation increases with pH and reflects a group with a pKa of 7.5. Inactivated enzyme is in the holo form with intact FMN. Four histidyls and a cysteinyl residue are modified by excess reagent. The restoration of enzymatic activity by hydroxylamine, the spectrophotometric and colorimetric amino acid analyses, and our previous studies on cysteine modification (Tsuge, H., and McCormick, D.B. (1979) in Flavins and Flavoproteins (Yamano, T., and Yagi, K., eds) Japan Scientific Societies Press, Tokyo, in press) all suggest that inactivation occurs solely by modification of histidine. Analyses by kinetic and statistical methods indicate that three histidines are modified slowly and are not critical for activity, while one histidine is modified nine times more rapidly and accounts for the observed inactivation. Inactivated enzyme shows no significant perturbations in structure, as evidenced by absorption, CD, fluorescence, and gel filtration, but is unable to bind the product, pyridoxal 5'-phosphate. Furthermore, the substrate-competitive inhibitor, pyridoxal 5'-phosphate oxime, protects from inactivation. Hence, diethylpyrocarbonate inactivates this enzyme by modifying a crucial histidyl residue at the substrate/product-binding site.     

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.