Properties of the Crystalline Quinolinate Phosphoribosyltransferase. from Hog Liver õ. Hiroshi TAGUCHI'* and Kazuo IwAt2*

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1 Agr. Biol. Chem., 39 (8), 1599 `1604, 1975 Properties of the Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver õ Hiroshi TAGUCHI'* and Kazuo IwAt2* Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 Received March 20, 1975 Quinolinate phosphoribosyltransferase has an important role in the NAD de novo biosynthetic pathway. Crystalline quinolinate phosphoribosyltransferase could be obtained for the first time from mammalian tissue. The crystalline enzyme preparation was certified to be homogeneous by polyacrylamide gel disc electrophoresis. Catalytic properties of this enzyme preparation were investigated. Optimum ph for the reaction was 6.1. Divalent cations were absolutely required and Mg2+ was the most effective. Michaelis constants for quinolinic acid and PRPP were 1.2 x 10-4 M and 1.8 x 10-4 M, respectively. Quinolinic acid could not be replaced by nicotinic acid or 2-amino nicotinic acid in this reaction. Di- and tri-valent cations fairly inhibited the reaction, but mono-valent cations had no effects. The reaction product was identified as S-nicotinic acid mononucleotide by its ultraviolet absorption spectra, paper chromatography, paper electrophoresis and its ORD spectrum. In all the NAD de novo pathways, quinolinic acid (pyridine-2,3-dicarboxylic acid) is a common intermediate and is converted enzymatically to NAD by three subsequent steps.'' The PRPP dependent conversion of quinolinic acid to nicotinic acid mononucleotide is the first step in the formation of nicotinic acidactive compounds. The enzyme which catalyzes this reaction is called quinolinate phosphoribosyltransferase (EC ). The enzyme has been purified from beef liver,"' Pseudomonad4' and the "Shiitake" mushroom (Lentinus edodes)5' and hog liver."' Crystallization from Pseudomonad4' and hog liver") only has been successful. The present paper describes the catalytic properties of the crystalline quinolinate phosphoribosyltransferase from hog liver. The purification, crystallization and physico-chemical properties of the enzyme from hog liver have been described in the preceding paper.7) õ The Biosynthesis of Nicotinamide Adenine Di nucleotide, Part V. For Part IV see the reference.1) Abbreviation used: NaMN, Nicotinic acid mono nucleotide. 1* Present address, Laboratory of Biochemistry, Faculty of Agriculture, Mie University, Tsu, Mie * Requests for reprints should be addressed to this author. MATERIALS AND METHODS Materials. Quinolinate phosphoribosyltransferase was purified from hog liver and twice crystallized by the methods described in the preceding paper.7' The enzyme decarboxylated 3.1 moles of quinolinic acid per hour per mg of protein under standard assay conditions. The labeled substrate of quinolinic acid- 2,3,7,814C was synthesized from aniline-u-14c and glycerol8' in the Daiichi Pure Chemicals Co., Ltd., Tokyo. PRPP (tetrasodium salt, purity 9095%) was purchased from Sigma Chemical Company. Authentic nicotinic acid mononucleotide (purity 100 %) was a gift from the Kyowa Hakko Kogyo Co., Ltd., Tokyo. Orotidine 5'-phosphate pyrophosphorylase and orotidine 5'-phosphate decarboxylase (mixed enzyme, crude powder from yeast) were obtained from Sigma Chemical Company. Methods. The standard assay system contained the following, in a total volume of 0.5 ml: Tris-maleate- NaOH buffer, ph 6.1 (25 pmoles); quinolinic acid- 2,3,7,8,14C (0.692 ymole, µci); PRPP (1 µmole); MgSO4 (0.5 µmole) and the enzyme (10 /cg). After incubating the assay mixture at 37 C for 30 min, the reaction was stopped by adding perchloric acid. By shaking the acidified mixture at 37 C for 90 min, the 14CO2 evolved was completely trapped by 9-phenylethylamine. Radioactivity was counted with a Packard Tri-Carb liquid scintillation spectrometer, model The amount of quinolinic acid utilized or of nicotinic acid mononucleotide formed by the enzymatic reaction is easily calculated by counting the evolved 14CO2. Details of the assay procedure are described in a

2 1600 H. TAGUCHI and K. IWAI previous paper 9) One unit of enzyme activity is defined as the amount of enzyme which decarboxylates 1mƒÊmole of quinolinic acid per hour under standard assay conditions. To identify the reaction product, large scale incubation was performed as follows: the reaction mixture contained Tris-maleate-NaOH buffer, ph 6.1 (100ƒÊmoles); quinolinic acid (3ƒÊpmoles); PRPP (4- ƒê moles); MgSO4 (2ƒÊmoles) and the enzyme (375 tog) TABLE I. METAL ION REQUIREMENTS FOR QUINOLINATE PHOSPHORIBOSYLTRANSFERASE ACTIVITY Standard assay conditions were used, except that the metal ions indicated were each added at 1mM. Ba, Hg, Ca andrb were added in the form of chlorides, Sr and Ag as nitrates and the other metals as sulfates. in a total volume of 2 ml. After incubating the mixture at 37 C for 2 hr, protein was removed by heating the whole in a boiling water bath for I min then cooling and centrifuging it at 10,000 x g for 15 min. Protein concentration was determined spectrophotometrically using an Ell of 9.96 at 280 run. Ultraviolet absorp tion spectra were measured with a Hitachi spectro photometer, model 124 connected to a Hitachi recorder, model QPD34. RESULTS Time course of the reaction Under standard assay conditions except for incubation time, the reaction proceeded linearly as a function of time for at least 50 min. The amounts of 14CO2 formed were propor tional to the amount of enzyme under standard assay conditions except for the enzyme concen tration. Effect of ph Maximum activity was attained at ph 6.1 and half maximum activities at ph 5.2 and 7.8 in Tris-maleate-NaOH buffer (Fig. 1). Metal ion requirement Divalent cations were absolutely required for the reaction as shown in Table I. Maximum activity was obtained with Mg2+ Magnesium ion was partially replaceable by Co2+, Cd2+, Zn2+, etc., but tri- and mono-valent cations were ineffective. The optimum concentration of Mg2+ was 1mM and the activity decreased at high concentrations (Fig. 2). FIG. 1. Effect of ph on Quinolinate Phosphoribosyl transferase Activity. Standard assay conditions were used, except for buffers at the ph values indicated, acetate buffer; œ- œ, Tris-maleate-NaOH buffer; -, Tris-HCl buffer; -, glycine-naoh buffer. FIG. 2. Effect of Magnesium Sulfate Concentration on Quinolinate Phosphoribosyltransferase Activity. Standard assay conditions were used, except for magnesium sulfate concentration as indicated.

3 Properties of Crystalline QA Phosphoribosyltransferase 1601 TABLE II. INHIBITION OF QUINOLINATE PHOSPHO RIBOSYLTRANSFERASE ACTIVITY BY METAL ION The metal ions listed were each added to the stand ard assay reaction mixture at a final concentration of I mat Hg, Ba, Ca and Rb were added in the form of chlorides, Ag and Sr as nitrates and the other metals as sulfates. The reaction was performed under standard assay conditions. FIG. 3. Effect of Quinolinic Acid Concentration Quinolinate Phosphoribosyltransferase Activity. Standard assay conditions were used, except for the quinolinic acid concentration as indicated. The Kin value was determined with a Lineweaver-Burk plot. on Effect of substrate concentration The quinolinic acid concentration was determined spectrophotometrically using a molar extinction coefficient of 4128 at 276 nm in aqueous solution and by the isotope dilution method. PRPP was determined by measuring the PRPP dependent decrease in the ultraviolet absorption of orotate at 295 nm according to the orotidine 5 L-phosphate pyrophosphorylase and orotidine 5 L-phosphate decarboxylase reactions.10) The Michaelis constants were determined with Lineweaver-Burk plots. The Km values for quinolinic acid and PRPP were 1.2 ~10-4M and 1.8 ~l0-4m, respectively (Figs. 3,4). Substrate specificity Though nicotinic acid, nicotinamide and 2- aminonicotinic acid were each added to the standard assay reaction mixture, they had no influence upon the activity. If they could combine with the active center of the enzyme, then inhibition of activity would occur. Ac cordingly quinolinic acid can't be replaced by them in the crystalline preparation. Inhibition by metal ions Metal ions were each added to the standard FIG. 4. Effect of PRPP Concentration on Quino linate Phosphoribosyltransferase Activity. Standard assay conditions were used, except for the PRPP concentration as indicated. The Km value was determined with a Lineweaver-Burk plot. assay reaction mixture at a final concentration of 1mM. Di- and tri-valent cations inhibited the reaction fairly well, while mono-valent cations had no effects except for Ag+ (Table II). Zn2+, Mn2+, Cd2+, Co2+ etc., which could

4 1602 H. TAGUCHI and K. IWAI partially replace Mg2+ as the metal ion require ment (Table I), strongly inhibited the reaction at 1 mm. But inhibition by Mg2+ was very weak in comparison with other di-valent cations when it was added at 1mM to the standard assay reaction mixture (i.e. the total concen tration of Mg2+ was 2mM). TABLE III. PAPER CHROMATOGRAPHY OF THE REACTION PRODUCT Paper chromatography of the peak I fraction was performed using the ascending method at 20 Ž with Whatman No. 1 filter paper and acidic, neutral and alkaline solvent systems. Paper electrophoresis was performed at 400 V/19 cm (6.5 ma) for 2.5 hr under cooling with ice. Identification of the reaction product Isolation of the product. The deproteinized A) 95% EtOH-1 M CHaCOONH4, ph 7.0 (5:2, v/v) B) n-buoh-ch3cooh-h2o (4:1:5, v/v) C) n-proh-nh4oh-h2o (11:7:2, v/v) D) Acetate buffer, ph 5.0; 400 V/19 cm, 6.5 ma, 2.5 hr. FIG. 5. Sephadex G-10 Column Chromatography of the Reaction Mixture. The deproteinized solution of the large scale incubation mixture (details described in METHODS) was charged on a Sephadex G-10 column (1.5 x 88 cm) then eluted with water at an elution speed of 16 ml per hour at room temperature (20 `25 Ž). Fractions of 1.5ml each were collected and the absorbance at 270nm was measured. Peak I, nicotinic acid mononucleotide; peak II, quinolinic acid; peak III, maleate in the buffer solution. reaction mixture of the large scale incubation (details are described in METHODS) was charged on a Sephadex G-10 column (1.5 ~ 88cm) and eluted with water at room tempera ture (20.25 Ž). Fractions of 1.5ml each were collected. Figure 5 shows the elution pattern. Three ultraviolet absorbing peaks were found; peaks I, II and III were identified as nicotinic acid mononucleotide, unreacted quinolinic acid and maleate in tris-maleate- NaOH buffer, respectively by comparing their ultraviolet absorption spectra and their Rf values on paper chromatography with those of authentic samples. The peak I fraction (fraction Nos. 50 and 51 in Fig. 5 were com bined) was verified as ƒà-nicotinic acid mono nucleotide by following methods: Ultraviolet absorption spectrum. The ultraviolet absorption spectrum of the peak I fraction is shown in Fig. 6 (solid line). Equal volumes of the peak I fraction and 2M KCN FIG. 6. Ultraviolet Absorption Spectra of the Reac tion Product. \, ultraviolet absorption spectrum of the peak I fraction (fraction Nos. 50 and 51 in Fig. 5 were combined); , ultraviolet absorption spectrum of a mixture of equal amounts of the peak I fraction and 2M KCN was measured. solution were mixed. After standing for 10- min at 20 Ž, the ultraviolet absorption spectrum of the mixture was measured (Fig. 6, dotted line). Both spectra agreed well with those of authentic sample. When the KCN solution was added to the peak I fraction,

Properties of Crystalline QA Phosphoribosyltransferase 1603 an absorption peak at 266 nm disappeared and a new peak at 317 nm appeared. This indi cates that nicotinic acid nucleotide was present.

5 Properties of Crystalline QA Phosphoribosyltransferase 1603 an absorption peak at 266 nm disappeared and a new peak at 317 nm appeared. This indi cates that nicotinic acid nucleotide was present. Paper chromatography and paper electro phoresis. Ascending paper chromatography was performed at 20 Ž using Whatman No. 1 filter paper in the following solvent system: 95 % ethanol-1 M ammonium acetate, ph 7.0 (5:2, v/v); n-butanol-acetic acid-water (4:1:5, v/v) and n-propanol-ammonia-water (11:7:2, v/v). Paper electrophoresis was carried out at 400 V/19 cm (6.5 ma) for 2.5 hr using acetate buffer, ph 5.0 under cooling with ice. The location of the compounds was detected with ultraviolet light, and fluorescence was confirmed after spraying KCN saturated methanol over the compound on the paper. Nicotinic acid nucleotide shows fluorescence by this treatment. The reaction product had the same behavior as the authentic nicotinic acid mononucleotide on paper chromatography and paper electrophoresis (Table III). ORD spectrum. Optical rotatory dis persion spectra of the peak I fraction were measured by an optical rotatory dispersion spectrometer, model ORD/UV-5, Japan Spec troscopic Co., Ltd. at 25 Ž. The spectrum corrected for the blank value is plotted in Fig. 7. The peak I fraction showed a negative Cotton effect around 250 nm. Hence, the compound in the fraction has the fi configuration.11) As demonstrated above, the peak I fraction was verified as ƒà-nicotinic acid mononucleotide. DISCUSSION Crystalline quinolinate phosphoribosyl transferase from mammals had never been obtained until the success of this study. Over all purification steps, the enzyme has been purified 5196-fold in an overall yield of 61 %. This crystalline preparation was certified to be homogeneous by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. No contamination by minor components in the crystalline preparation was detected. The radioassay for quinolinate phospho ribosyltransferase using quinolinic acid-2,3,7,8-14c as the substrate is very useful because of its high specificity, conveniency and accuracy. At the 2,3 and 8-positions of quinolinic acid, [14C] is not required, only [7-14C] is essential for this radioassay. However, with com mercially obtainable labeled precursor and organic synthesis method used, [14C] is located at the 7-position and also the 2,3 and 8-positions of quinolinic acid. In general the catalytic properties of this crystalline enzyme resemble many of the characteristics reported in the data for the liver2,3) and Pseudomonad4) enzymes. For example, the optimum ph values for the reac tion were all about neutral (6.1 `7.1). The Km values for quinolinic acid and PRPP were also relatively alike, both being about the order of 10-4M. Di-valent cations were required for the reaction and Mg2+ was most effective (optimum concentration were around 10-3M) in all the enzyme preparations. The reaction product was identified as ƒànicotinic acid mononucleotide by several methods. Biologically active NAD as the FIG. 7. Optical Rotatory Dispersion Curve of the Reaction Product. Optical rotatory dispersion spectra of the peak I fraction were measured with an optical rotatory dis persion spectrometer, model ORD/UV-5, Japan Spectroscopic Co., Ltd. at 25 Ž. The spectrum cor rected for the blank value is shown. coenzyme has ƒà-form. The existence of a- NAD in animals and microorganisms has been reported,12,13) but it is believed that this a- NAD has unknown important biological functions or is merely an artifact which occurs during isolation. No role for a-nad in

6 1604 H. TAGUCHI and K. IWAI organisms has been shown, and at which step of the NAD biosynthetic pathway it is synthe sized is unknown. The ƒ -form in the pyridine ring and ribose bonding is most likely to form at the nucleotide forming reaction step, i.e. the quinolinate phosphoribosyltransferase catalyzing reaction. But in the hog liver enzyme the ƒà-form alone was formed. In mammals NAD is derived from both nicotinic acid (or nicotinamide) and trypto phan. Quinolinic acid is an intermediate in the tryptophan-nad pathway. Nicotinic acid mononucleotide is formed from nicotinic acid and PRPP by the reaction of nicotinate phosphoribosyltransferase (EC ). In this reaction ATP has an interesting role."' The crystalline quinolinate phosphoribosyl transferase from hog liver lacked nicotinate phosphoribosyltransferase activity. The enzymatic formation of fl-nicotinic acid mononucleotide from quinolinic acid in hog liver was confirmed using the crystalline enzyme preparation. Acknowledgement. We are grateful to the late Prof. T. Mitsui and Dr. H. Oohigashi of the Depart ment of Food Science and Technology, the Faculty of Agriculture, Kyoto University, for measuring the ORD spectra. REFERENCES 1) R. K. Gholson, Nature, 212, 933 (1966). 2) S. Nakamura, M. Ikeda, H. Tsuji, Y. Nishizuka and O.Hayaishi, Biochem. Biophys. Res. Commun., 13, 285 (1963). 3) R. K. Gholson, I. Ueda, N. Ogasawara and L. M. Henderson, J. Biol. Chem., 239, 1208 (1964). 4) P. M. Packman and W. B. Jakoby, ibid., 240, PC 4107 (1965). 5) H. Taguchi and K. Iwai, J. Nutr. Sci. Vitaminol., 20, 269 (1974). 6) K. Iwai and H. Taguchi, Biochem. Biophys. Res. Commun., 56, 884 (1974). 7) H. Taguchi and K. Iwai, Agr. Biol. Chem., 39,1493 (1970).8) "Methods in Enzymology," Vol. XVII A, ed. by H. Tabor and C. W. Tabor, Academic Press Inc., New York, N. Y., 1970, p ) K. Iwai and H. Taguchi, J. Nutr. Sci. Vitaminol., 19, 491 (1973). 10) A. Kornberg, I. Lieberman and E. S. Simms, J. Biol. Chem., 215, 389 (1955). 11) T. Imai and S. Suzuki, Seikagaku, 38, 618 (1966). 12) N. O. Kaplan, M. M. Ciotti, F. E. Stolzenbach and N. R. Bacher, J. Am. Chem. Soc.. 77, 815 (1955). 13) S. Suzuki, K. Suzuki, T. Imai, N. Suzuki and S. Okuda, J. Biol. Chem., 240, PC 554 (1965). 14) L. D. Smith and R. K. Gholson, ibid., 244, 68 (1969).

Enzymatic Assay of NAD-PYROPHOSPHORYLASE (EC )

Enzymatic Assay of NAD-PYROPHOSPHORYLASE PRINCIPLE: ß-NMN + ATP NAD-Pyrophosphorylase > ß-NAD + PP ß-NAD + Ethanol ADH > ß-NADH + Acetaldehyde Abbreviations used: ATP = Adenosine 5'-Triphosphate ADH =