[48] 6-Phospho-d -gluconate dehydrogenase from sheep liver

214

OXIDATION--REDUCTION ENZYMES

[48]

TABLE III AMINO ACID COMPOSITION OF HUMAN ERYTHROCYTE GLUCOSE-6-PHOSPHATE DEHYDROGENASE

Amino acid

Residues per subunit (molecular weight 53,000)

Aspartie acid Threoniae a Serine ~ Glutamic acid Proline Glycine Alanine Valine b Methionine Isoleucine b Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine ~ Tryptophan ~ Total

49.2 19.4 23.9 55.5 22.8 32.6 29.8 31.5 12.0 25.0 42.2 17.0 23.1 11.5 27.3 28.1 7.2 6.9 465.0

" Extrapolated to zero time from 24, 48, and 72-hr hydrolyses. .bFinal value after 72-hr hydrolysis. Determined as cysteic acid. d Determined by the method of W. L. Bencze and K. Schmid, Anal. Chem. 9.9, 1193 (1957).

[48]

6-Phospho-D-gluconate Dehydrogenase from Sheep Liver B y £-~ICHAEL SILVERBERG a n d KEITH DALZIEL

6-Phospho-D-gluconate -{- N A D P ~ D-ribulose 5-phosphate + CO2 T N A D P H

6-Phosphogluconate dehydrogenase was first highly purified from a mammalian tissue by Villet and Dalziel, 1 who obtained an apparently homogeneous preparation from sheep liver. The isolation procedure described here is an extensive modification 2 of that method, which gives a preparation of higher specific activity from which the enzyme has been 1R. H. Villet and K. Dalziel, Biochem. J. 115, 639 (1969). M. Silverberg and K. Dalziel, Eur. J. Biochem. 38, 229 (1973).

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215

cryst'dlized. Other modifications have been published recently for the isolation of the enzyme from sheep liver '~ and rat liver2

Assay Method

Reagents Glycine-NaOH buffer, 0.17 M, pH 8.9 MgC1..,, 0.1 M Sodium 6-phosphogluconate, 20 mM NADP, 2 mM

Procedure. The initial rate of reduction of NADP at 25 ° is determined by absorbance measurements at 340 nm with a reaction mixture comprising 1.8 ml of buffer, 0.6 ml of MgCI.., 0.3 ml of 6-phosphogluconate and 0.3 ml of NADP, reaction being initiated by the addition of 5 or 10 ~1 of enzyme solution containing about 0.15 t~g of enzyme. A unit of enzyme is that amount which causes an initial rate of formation of 1 ~mole of NADPH per minute. Determination of Protein and Specific Activity. The specific activity (units per milligram of protein) was calculated from protein determinations by the method of Warburg and Christian 5 in the tissue extract after acidification and at subsequent stages of the purification procedure from absorbance measurements at 280 rim, assuming ~l~'l~m = 10.0, on which basis the specific activity of the pure enzyme is 19. From dry weight determinations, 2 the extinction coefficient of the pure enzyme at 280 nm is ~,~'1~,~ = 11.4, and on this basis the specific activity is 21. Purification Procedure All operations are performed at 0-5 °, and all buffers are made up in glass-distilled water and contain 1 mM EDTA. Step 1. Extraction and Acid Treatment. Fresh, minced sheep liver, 800 g, is soaked for 20 hr in 1.6 liters of 0.125 M potassium phosphate buffer, pH 7.0, and filtered through muslin. The filtered extract is brought to pH 5.0 by the gradual addition of 20% acetic acid and, after standing for 10 min, is centrifuged at 14,000 g for 45 rain. The decanted supernatan't is immediately brought to pH 6.0 with 25% ammonia solution. The acid treatmen~ results in a 2- to 3-fold increase of specific activity, with *J. E. D. Dyson, R. E. D'Orazio, and W. H. Hanson, Arch. Biochem. Biophys. 154, 623 (1973). 4D. Procsal and D. Holten, Biochemistry 11, 1310 (1972). 50. Warburg and W. Christian, Biochem. Z. 310, 384 (1942).

216

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[48]

a yield of 90% if carefully carried out. However, the initial extract before acid treatment is difficult to clarify by centrifugation and assays at this stage are not reliable. Values for the yield in the subsequent stages are therefore referred to the supernatant from the acid treatment. Step 2. Ammonium Sul]ate Fractionation at pH 6.0. Ammonium sulfate (analytical grade) is added piecemeal to the stirred supernatant from step 1, to a final concentration of 300 g/liter. The pH is monitored and kept at 6.0 by addition of 25% ammonia solution. After 1.5 hr the precipitate is removed by centrifugation at 14,000 g for 40 min and additional 90 g of ammonium sulfate per liter is added to the supernatant. The solution is left overnight, and the precipitate is collected by centrifugation as before. The precipitate is resuspended in the minimum volume of 0.125 M phosphate buffer, pH 7.0, containing 1 mM 2-mercaptoethanol. The suspension is dialyzed against 10 mM phosphate buffer, pH 7.0, containing 1 mM mercaptoethanol for about 30 rain, to decrease the salt concentration and dissolve the protein without a large increase in volume. Step 3. Gel Filtration. The protein solution is ehromatographed on a column of Sephadex G-100 (4.5 cm diameter )< 90 cm) equilibrated with 10 mM phosphate buffer, pH 7.0, containing 1 mM 2-mercaptoethanol. The protein is eluted with the same buffer at a flow rate of 30 ml/hr. The enzyme-containing fractions are pooled and brought to pH 8.0 with 25% ammonia solution. Step 4. Chromatography on DEAE-Sephadex. The product from the previous step is loaded onto a column (3.75 cm diameter X 20 em) of Sephadex A-50 equilibrated with 10 mM phosphate buffer, pH 8.0. The enzyme is eluted at a flow rate of 30 ml/hour with a concave gradient made from 1 liter of 35 mM phosphate buffer and 1 liter of 10 mM phosphate buffer, pH 8.0. The position of elution of the enzyme in the gradient is somewhat variable, presumably because of the difficulty of exactly reproducing the properties of the ion-exchanger in dilute phosphate buffer. Step 5. Ammonium SulJate Fractionation at pH 8.0. The pooled enzyme-eontaining fractions from the previous step are concentrated by ultrafiltration under oxygen-free nitrogen (Amicon system, membrane PM30) until the enzyme concentration is 1.5 units/ml. The latter value is fairly critical; with smaller values, the yield in the ammonium sulfate fractionation is low, and with higher values colored impurity is not completely removed in either this or the following step. Ammonium sulfate is added to a concentration of 350 g/liter and the precipitate is removed after 30 rain by centrifugation at 23,000 g for 30 rain. A further 30 g/liter ammonium sulfate is added, and the suspension is left overnight. The precipitate is removed as before and 100 g of ammonium sulfate per liter

[48]

6-PHOSPHOGLUCONATE DEHYDROGENASE

217

is added to the supernatant. After 48 hr, the enzyme-containing precipitate is isolated by centrifugation and dissolved in 0.1 M potassium phosphate buffer, pH 7.0, to give a total volume of 10 ml. Step 6. Chromatography on CM-Sephadex. The solution is passed through a column of Sephadex G-25 (2.5 cm diameter X 40 cm) equilibrated with 10 m M phosphate buffer, pH 7.0, containing 50 m M KC1. The eluate is loaded onto a column of CM-Sephadex (C-50) of the same dimensions and preequilibrated with the same buffer-KC1 solution as the G-25. The enzyme is eluted with a linear gradient of 1 liter of 0.25 M KC1 into 1 liter of 0.05 M KC1 in 10 m M phosphate buffer, pH 7.0, at a flow rate of 60 ml/hr. The eluates containing the pure enzyme are concentrated by ultrafiltration with the addition of 0.125 M phosphate buffer, p H 7.0. A small precipitate sometimes forms during this process, and the specific activity increases slightly, presumably indicating removal of inactive and denatured enzyme. The product may be stored for some weeks at 2 ° without loss of activity. The results obtained in a typical preparation are given in the table. The isolation procedure has been scaled up to 10 kg tissue without significant modification. Crystallization. Large crystals are readily obtained 2 by dialysis of the pure enzyme solution, 8 mg/ml in 50 m M phosphate buffer, p H 6.2-7.8, against 55% saturated ammonium sulfate solution for 2 days. The specific activity of the redissolved crystalline enzyme is usually about, 20% smaller than that of the original enzyme preparation. Crystals over 1 mm long, suitable for X - r a y diffraction studies, have also been obtained2, ~ SUMMARY OF PURIFICATION PROCEDURE

Step 1. Acidified extract 2. (NH4)~SO4, pH 6.0

3. 4. 5. 6.

Sephadex G-100 DEAE-Sephadex (NH4)2SO4,pH 8.0 CM-Sephadex, ultrafiltration

Volume (ml)

Protein (g)

1430 70 440 500 10 3

42 10.5 5.3 0.64 0.22 0.030

Specific Activity activity (units/ml) (units/mg) 1.32 16.6 2.75 1.72 80.4 185.0

0. 045 0.110 0.23 1.35 3.68 18.7

Yield (%) 100 62 64 46 43 29

e M. Silverberg, K. Dalziel, and M. J. Adams, Biochem. Soc. Trans. 1, 1132 (1973). 7M. J. Adams, unpublished work, 1973.

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OXIDATION--REDUCTION ENZYMES

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Properties

Chemical and Physical. The enzyme does not contain significant amounts of Zn 2+, 5'fn ~+, or Mg ~+, nor are added bivalent metal ions essential for activity, s The complete amino acid composition has been reported, and of the seven thiol groups per subunit two react rapidly with Ellman's reagent and p-hydroxymercuribenzoate and appear to be essential for activity. ~ The extinction coefficient, for the protein at 280 nm calculated from the tryptophan and tyrosine contents (7 and 11 per subunit, respectively) is ~l~l~cm= 11.4, identical with that estimated directly from dry weight determinations. 2 The partial specific volume calculated from the amino acid composition is 0.741. The molecular weight from gel filtration, polyacrylamide gel electrophoresis, and sedimentation equilibrium studies by the meniscus depletion method is 94,000 ± 2000, and there are two subunits in the molecule as shown by SDS gel electrophoresis and studies of NADPH bindingY Similar molecular weights and subunit composition have been reported for the enzymes isolated from Candida utilis, 9 Bacillus stearothermophilus, 1° rat liver ~ and human erythrocytes.'1 The higher value of 129,000 for the molecular weight of the sheep liver enzyme earlier reported by Villet and Dalziel 1 must be attributed to the presence of impurities. Preliminary Crystallographic Data. X-Ray diffraction studies of the enzyme crystals by Adams 6,7 show the crystals to be face-centered orthorhombic, space group C2221. The asymmetric unit is probably the subunit (47,000 daltons), of which there are 8 in the unit cell of dimensions 72.7 A X 149.0 A X 103.8 A. Thermodynamics. The enzyme-catalyzed reaction is readily reversible. Dissolved COs, not bicarbonate ion, is the immediate substrate or product. :2 The equilibrium constant for the reaction N A D P -4- 6-phosphogluconate ~ N A D P H + ribulose 5-phosphate + CO2 (gas)

(1)

is K, = 2.62 atmospheres, at 25 °, and is independent of pH and ionic strength? s This refers to a standard state of 1 arm pressure of COs. For a standard state of the ideal molar solution of COz, the equilibrium constant is Kc = 79 mM. For reaction (1), AG ° at 25 ° is --0.51 kcal/mole, and AH ° is 13.75 kcal/mole. Coenzyme. The enzyme from sheep liver does not utilize NAD as coR. H. Villet and K. Dalziel, Eur. J. Biochem. 27, 251 (1972). 9 M. Rippa, M. Signorini. and S. Pontremoli, Eur. J. Biochem. 1, 170 (1967). ~oB. M. F. Pearse, unpublished work, 1973. 1~B. M. F. Pearse and M. A. Rosemeyer, this volume [49]. ~R. H. Villet and K. Dalziel, Biochem. J. 115, 633o(1969)

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219

enzyme.13 The binding of the active coenzyme NADPH to the enzyme results in increased nucleotide fluorescence, s,18 which is further augmented in the presence of 6-phosphogluconate, indicating the formation of an abortive ternary complex. From fluorescence titrations, 13 the presence of one binding site for NADPH on each of the two subunits of the enzyme has been established, and the dissociation constant of the binary enzyme-NADPH complex is 5.7 ~M in 0.1 M phosphate buffer, pH 7.0, at 25 °. From kinetic studies 13 of the inhibition of the oxidative decarboxylation reaction by NADPH, Ki is 9 ~M. From the initial rate parameters for the oxidative decarboxylation reaction at pH 7.0, dissociation constants for the enzyme-NAD compound are estimated indirectly to be 2.8 ~M in 0.13 M triethanolamine buffer and 26 ~M in 0.1 M phosphate buffers Kinetic Constants. For the oxidative decarboxylation reaction in glycine buffer,s with saturating substrate concentrations, the pH optimum is 8.9. In Tris-acetate buffer, the pH optimum was found to increase with increasing substrate concentrations up to pH 9.1, above which the enzyme was unstable2 In 0.13 M triethanolamine buffer, pH 7.0, at 25 °, Km values, with saturating concentrations of the other substrate(s), are s.1, 6.8 ~M for NADP 6.9 ~M for 6-phosphogluconate, 0.22 ~M for NADPH, 20 ~M for ribulose 5-phosphate and 34 mM for dissolved CO._,. In 0.1 M phosphate buffer, pH 7.0, at 25 °, K., values are 30 ~M for NADP and 290 ~M for 6-phosphogluconate, in both cases considerably larger than in triethanolamine buffer. The maximum specific rate is also 6 times greater in phosphate than in triethanolamine,s however, so that phosphate does not simply act as a competitive inhibitor with respect to NADP and 6-phosphogluconate. Also in 0.1 M phosphate buffer, the effect of 20 mM Mg ~÷on the initial rate parameters for the oxidative deamination is small, the main effect being a small decrease of the K~,~for NADP, while higher Mg 2÷ concentrations cause a small inhibition, s At pH 7.7 and 30 °, in 50 mM Tris-acetate-KC1 buffer, ionic strength 0.1, Km values are 6.8 ~M for NADP and 16 ~tM for 6-phosphogluconate2 In this medium the addition of 20 mM Mg 2÷ causes inhibition by a 3-fold increase of the K,,, for 6-phosphogluconate, while at lower ionic strengths and Mg '-'+ concentrations, there is a small activation; it appears that the relatively small effects of increasing ionic strength, Mg 2÷, Ca ~+, and Mn 2÷ ion concentrations are similar and interchangeable, and mainly due to effects on the Km for 6-phosphogluconate2 13 M. Silverberg, D. Phil. Thesis, Oxford University, 1973. 14R. H. Villett and K. Dalziel, Eur. J. Biochem. 27, 244 (1972).

220

OXIDATION--REDUCTION ENZYMES

[49]

Initial rate parameters for the oxidative decarboxylation reaction have been estimated at other pH values also. :~,~ From the effects of temperature on the maximum rate at pH 7.7, the activation energy ~ is 15.5 kcal/mole. Inhibitors. Dyson and D'Orazio 15,1'~ have studied a range of potential metabolic inhibitors. Nucleoside 5'-triphosphates and 5'-diphosphates inhibit competitively with respect to both N A D P and 6-phosphogluconate, with Ki values ranging from 0.2 m M for G T P to 1.3 m M for A T P in Tris-acetate buffer, pH 7.0, at 30 °. The 5'-monophosphates and 3'-monophosphates have larger Ki values (2-11 mM). For 2'-AMP and 2'-GMP, the Ki values are 0.36 m M and 0.57 mM, respectively. Fructose-l,6-diphosphate is a strong inhibitor (K~ = 0.07 mM), the effect of which is counteracted by EDTA. Other inhibitors with Ki values in the range 3-9 m M include fructose 1-phosphate, fructose 6-phosphate, glucose 6-phosphate, orthophosphate, pyrophosphate, oxaloacetate, and citrate. The sugar phosphates are competitive with respect to 6-phosphogluconate and noncompetitive with respect to NADP. 15j. E. D. Dyson and R. E. D'Orazio, J Biol. Chem. 248, 5428 (1973). l~J. E. D. Dyson and R. E. D'Orazio, Biochem. Biophys. Res. Commun. 43, 183 (1971).

[49] 6-Phosphogluconate Dehydrogenase from H u m a n Erythrocytes By BARBARAM. F. PEARSEand MICHAELA. ROSEMEYER 6-Phosphogluconate -t- NADP ~ ribulose 5-phosphate ~- CO2 ~c NADPH The details of the following purification of 6-phosphogluconate dehydrogenase [6-phospho-D-gluconate:NADP + oxidoreductase (decarboxylating), EC 1.1.1.44] have been reported in the E u r o p e a n Journal oJ Biochemistry. 1 The subunit structure of the enzyme has been reported in the same journal. ~

Enzyme Assay Principle. The activity of 6-phosphogluconate dehydrogenase is measured in the presence of excess 6-phcsphogluconate and N A D P by the

B. M. F* Pearse and M. A. Rosemeyer, Eur. J. Biochem. 42, 213 (1974). 2B. M. F. Pearse and M. A. Rosemeyer, Eur. g. Biochem. 42, 225 (1974). 1