Purification and properties of coproporphyrinogen III oxidase from bovine liver

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COPROPORPHYRINOGEN I I I OXIDASE FROM BOVINE LIVER

355

enzyme is also inhibited by diethyl pyrocarbonate (DEPC). This inhibition is reversed by hydroxylamine, indicating reaction of DEPC with at least one histidine residue, but is not prevented by prior incubation with substrate. Inhibition by metals has been studied because iron is involved in the pathogenesis of the human U R O D deficiency disorder, porphyria cutanea tarda. In addition t o H g 2+, human U R O D is inhibited by Cu 2+ and P t 2+ but not by Co 2+, M g 2+, M n 2+, Pb 2+, o r S b 3+. Although inhibition of partially purified human enzyme by iron has been reported, neither F e z+ n o r Fe 3+ appears to inhibit purified human UROD. s

[41] P u r i f i c a t i o n a n d P r o p e r t i e s o f C o p r o p o r p h y r i n o g e n III O x i d a s e f r o m B o v i n e L i v e r

By T A K E O

YOSHINAGA

Introduction Coproporphyrinogen oxidase (CPO, EC 1.3.3.3), the sixth enzyme of the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX by oxidative decarboxylation of two propionate groups at positions 2 and 4 of the tetrapyrrole to yield vinyl groups.1 3 The mammalian enzyme is located in the intermembrane space of mitochondria and is soluble or very loosely bound to the membrane*,5; in contrast, CPO in yeast is cytosolic.6 Since 19611 there have been many attempts to purify the enzyme. Yoshinaga and Sano succeeded in obtaining a preparation from bovine liver that was almost pure 7 and CPOs from yeast 6 and mouse liver 8 were also purified. However, some discrepancies in molecular properties of these enzymes were seen; for example, the molecular mass of bovine liver CPO was 71.6 kDa in contrast with CPO of mouse or yeast, which was 35 kDa. A modified purification procedure of bovine

a S. Sano and S. Granick, J. BioL Chem. 236, 1173 (1961). 2 A. M. del C. Batlle, A. Benson, and C. Rimington, Biochem. Z 97, 731 (1965). 3 H. G. Elder, J. O. Evans, J. R. Jackson, and A. H. Jackson, Biochem. J. 169, 215 (1978). 4 H. G. Elder and J. O. Evans, Biochem. J. 172, 345 (1978). 5 B. Grandchamp, N. Phung, and Y. Nordmann, Biochem. J. 176, 97 (1978). 6 j. M. Camadro, H. Chambon, J. Jolles, and P. Labbe, Eur. J. Biochem. 156, 579 (1986). 7 T. Yoshinaga and S. Sano, J. Biol. Chem. 255, 4722 (1980). s M. Bogard, J. M. Camadro, Y. Nordmann, and P. Labbe, Eur. J. Biochem. 181, 417 (1989).

METHODS IN ENZYMOLOGY, VOL. 281

Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25

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liver has permitted us to clone the enzymes from mouse 9 and from human, m The analysis of these cDNAs provided a molecular mass of 37 kDa from amino acid compositions. Coproporphyrinogen oxidase genes from Saccharomyces cerevisiae, u Salmonella typhimurium, 12 Escherichia coli, 13 and soybean TM have been also isolated and sequenced. Recombinant mouse CPO expressed in E. coli has a specific activity that is almost equivalent to that of the bovine liver enzyme, and contains copper responsible for the enzyme activity.15 Site-directed mutagenesis of highly conserved His-158 caused the complete loss of the activity with concomitant release of bound copperJ 5 This chapter describes an improved method of purification of CPO from bovine liver, purification of recombinant mouse CPO from E. coli, properties of the enzymes, and results of point mutation of histidine residues. Purification of Coproporphyrinogen Oxidase Enzyme Assay Methods to assay CPO are separated into two groups. The first utilizes the absorbance of protoporphyrin after extraction from the organic phase using different concentrations of HCI 1 and the second method uses radioactivity in the determination of the separated protoporphyrin. 16 Separation of protoporphyrin from coproporphyrin and determination by high-performance liquid chromatography (HPLC) equipped with a reversed-phase column and fluorometric monitor can be usedJ 7 The assay method described here can be used to treat many samples at the same time. Coproporphyrinogen III as the substrate is prepared by hydrolysis of coproporphyrin tetramethyl ester with 6 N HCI for 6 hr at room temperature and pooled in 0.01 N NaOH in the dark at a concentration of 10 mM. Just 9 H. Kohno, T. Furikawa, Y. Yoshinaga, R. Tokunaga, and S. Taketani, J. Biol. Chem. 268, 21359 (1993). 10 S. Takenani, H. Kohno, T. Furukawa, T. Yoshinaga, and R. Tokunaga, Biochim. Biophys. Acta 1183, 547 (1994). 11 M. Zagorec, L.-M. Buhler, I. Treich, T. Keng, L. Guarente, and R. Labbe-Bios, J. Biol. Chem. 263, 9718 (1988). 12 K. Xu and T. Elliott, J. Bacteriol. 175, 4990 (1993). 13 B. Troup, M. Jahn, C. Hungerer, and D. Jahn, J. Bacteriol. 1"/6, 673 (1994). 14 O. Madsen, L. Sandal, N. N. Sandal, and K. A. Macker, Plant Mol. Biol. 23, 35 (1993). 15 H. Kohno, T. Furukawa, R. Tokunaga, S. Taketani, and T. Yoshinaga, Biochim. Biophys. Acta 1292, 156 (1996). 16B. Grandchamp and Y. Nordmann, Enzyme 28, 196 (1982). 17 R~ Guo, C. K. Lira, and T. J. Peter, Clin. Chim. Acta 177, 245 (1988).

[41]

COPROPORPHYRINOGEN I I I OXIDASE F~OM BOVINE LIVER

357

before use, coproporphyrin is quickly reduced by granular 3% (w/v) sodium amalgam (1 g/ml) in the presence of 0.1 M 2-mercaptoethanol with vigorous shaking. When the solution becomes colorless or pale yellow, it is drawn through a small cotton ball with a pipette and transferred to a new test tube. The reduced solution is neutralized with 6 N H3PO4, and then diluted with 4 vol of ice-cooled 0.1 M Tris-HCl buffer, pH 7.6. Reaction mixtures contain 5-100/zl of enzyme solution, 100/zM coproporphyrinogen, and 20 mM 2-mercaptoethanol in 80-100 mM Tris-HC1 buffer, pH 7.6, at a final volume of 0.5-0.1 ml in 10-ml glass tubes. The mixtures are incubated at 37° for 15-60 min in the dark. After incubation, 4 ml of acetic acid-ethyl acetate (1 : 3, v/v) is added followed by exposure to strong light (500-W tungsten light) for 5 to 30 min; the color of the mixture becomes pinkish orange and then the color diminishes. Two milliliters of H20 is added followed by stirring with a Vortex mixer for 5 sec. After the mixture separates into organic and aqueous phases, the aqueous phase is removed with a long 18-gauge needle connected to an aspirator through a stop cock or to a cylinder. One milliliter of 2.5 N HCI is added to the organic phase, which is then vortexed for 5 sec; porphyrins are extracted into the aqueous phase and then the organic phase is removed. The pH of the aqueous phase is brought to pH 3.5 by addition of 1.6 ml of saturated sodium acetate. Four milliliters of ethyl ether is added to each tube and porphyrins are extracted into the organic phase. The aqueous phase is then aspirated off. Unreacted coproporphyrin is removed by washing the organic phase with 2 ml of 0.12 N HC1 three or four times. The amount of remaining substrate can be determined by measuring the absorbance of the washing solution at 402 nm (EmM = 470 M 1 cm-l). Protoporphyrin is extracted with 1.0 or 2.0 ml of 3 N HC1 from the organic phase and the amount is determined by measuring absorbance at 408 nm (Emit = 262 M -1 cm-1). This method is convenient as only one tube is necessary for each sample throughout the procedure from incubation to product extraction. Tenmilliliter conical centrifuge tubes with screw caps are recommended to prevent overflow of contents on vortexing. One unit of CPO is defined as the amount that catalyzes the conversion of 1 nmol of protoporphyrinogen per hour at 37°.

Preparation of Crude Enzyme Source In mammalian cells CPO is located in the intermembrane space of mitochondria,4'5 and is almost soluble or very loosely bound to the membrane. When the mitochondrial fraction is prepared carefully from fresh tissue to obtain an intact preparation, the specific activity of CPO is higher among the other fractions; however, the total amount of activity in these

358

HEME

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other fractions is not large. If a large column (62 x 900 mm) is available, it is not important to start with pure mitochondria. All the subsequent operations are carried out at temperatures between 0 and 4°. One kilogram of fresh bovine liver (loss of the enzyme activity is less than 50% over 1 month when fresh liver is stored at - 8 0 °) is sliced and homogenized in 4 liters of 20 mM Tris-HC1 buffer, pH 7.6, containing 20 mM 2-mercaptoethanol and 0.2 mM phenylmethylsulfonyl fluoride (TMP buffer), with a Waring blender under cooling. The homogenate is centrifuged at 15,000 g for 20 rain and the supernatant is collected. Ammonium Sulfate Fractionation. Powdered ammonium sulfate is added to the supernatant to 35% saturation, the pH of the mixture is adjusted to pH 7.8, and the solution is stirred for 30 rain. After removal of the precipitate by centrifugation at 15,000 g for 30 rain, ammonium sulfate is added to the solution to 45% saturation. The mixture is stirred for 30 min, then the enzyme is precipitated by centrifugation at 15,000 g for 30 min. The activity of CPO in the precipitated state is not stable, so the precipitate obtained is dissolved with a minimum volume of TMP buffer and dialyzed twice against 20-50 vol of TMP buffer for 6 hr.

Purification of Enzyme DEAE-Cellulofine Column Chromatography. Dialyzed crude enzyme is centrifuged at 30,000 g for 20 min to remove insoluble materials, followed by loading onto a column of DEAE-Cellulofine (62 x 900 cm) (SeikagakuKogyo, Tokyo, Japan) preequilibrated with TMP buffer at a flow rate of 100 ml/hr. The procedure for preparation of the column is important to obtain high resolution, especially when using a large column. No air bubbles should be trapped in the column. The column is washed with 0.9 liter of TMP buffer followed by elution of the enzyme with a linear gradient of NaCI from 0 to 0.5 Min 5 liters of TMP buffer at a flow rate of 200 ml/hr. Thirty-milliliter fractions are collected. Typical elution patterns of protein and CPO activity are shown in Fig. 1. Coproporphyrinogen oxidase is eluted at 0.i M NaC1. Fractions with CPO activity over 50% of peak activity are pooled and the volume ratio of this pooled fraction to total eluate is 6-7%. Hydroxylapatite Column Chromatography. The pH of the combined fractions is adjusted to pH 6.5 with H3PO4. Coproporphyrinogen oxidase is not stable under acidic conditions, and thus care must be taken to avoid dropping below pH 6.5. The fractions are loaded on a column of hydroxylapatite (26 × 300 mm) pretreated with 20 mM potassium phosphate buffer, pH 6.5, containing 10 mM 2-mercaptoethanol and 0.2 mM phenylmethylsulfonyl fluoride (PMP buffer) at a flow rate of 50 ml/hr. Hydroxylapatite gel is usually handled with phosphate buffer, because the gel is protected from

[41]

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FIG. 1. Elution profile of DEAE-Cellulofine column chromatographyfor purification of CPO from bovine liver. Details are described in text. Fraction 1 indicates the starting position of the gradient for elution. The CPO activities in the fractions are shown with a dashed line and absorbance of eluate monitored at 260 nm is shown with a solid line. The scales of these parameters are expressed in arbitrary units on the y axis. The concentrations of NaC1 in the fractions are shown with a dotted line. dissolving by phosphate ions. In this case, however, the fractions are loaded onto the column in Tris-HC1 buffer from the previous step, which exposes the flesh surface of the gel and promotes binding of CPO. The column is washed with 200 ml of PMP buffer at a flow rate of 70 ml/hr. The enzyme is eluted with a linear gradient of 500 ml of 20 m M PMP buffer to 500 ml of 300 m M potassium phosphate buffer, p H 7.6, containing 10 mM 2-mercaptoethanol and 0.2 m M phenylmethylsulfonyl fluoride at a flow rate of 70 ml/hr. The eluate is collected in 10-ml fractions. CPO was eluted at a phosphate concentration of 110 raM. The elution position of CPO is variable because the performance of hydroxylapatite differs between preparations, manufacturers, and even within the same batch. Hydroxylapatite for column chromatography from Nacalai Tesque (Kyoto, Japan) is sold as a dried powder, is easy to handle, and has good performance with regard to both capacity and flow rate. Fractions with CPO activity over 50% of peak activity are combined and 13 m M 2-mercaptoethanol and solid ammonium sulfate up to 1.0 M are added. The volume of combined fractions is about 15% of that of total eluate. Hydrophobic Column Chromatography by Butyl-Toyopearl. Fractions with increased ionic strength are loaded onto a column (26 × 250 mm) of butyl-Toyopearl (TSK gel butyl-Toyopearl 650S; Tosoh Corporation, Tokyo, Japan) preequilibrated with TMP buffer containing 1.0 M ammonium sulfate at a flow rate of 30 ml/hr. The column is washed with TMP buffer containing 0.8 M ammonium sulfate at a flow rate of 50 ml/hr until

360

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the absorbance monitored at 260 nm returns to the initial level. The enzyme is eluted with a linear gradient of ammonium sulfate from 0.8 to 0 M in 600 ml of TMP buffer at a flow rate of 40 ml/hr and the eluate is collected in 6-ml fractions. Fractions with CPO activity over 60% of peak activity are combined. The peak of activity is eluted at a position corresponding to 0.12 M ammonium sulfate and the volume of fractions combined is about 13% of that of the total eluate. Coproporphyrinogen oxidase in the fractions is collected by precipitation using 60% saturation of ammonium sulfate. The enzyme pellet is dissolved in 2.0 ml of 50 mM Tris-HCl buffer containing 10 mM 2-mercaptoethanol. Cellulofine GCL-2OOOm Column Chromatography. After removal of insoluble materials by centrifugation at 30,000 g for 20 min, the concentrated CPO is loaded on a column for gel filtration with Cellulofine GCL-2000m (21 x 1100 mm) (Seikagaku-Kogyo, Tokyo, Japan) preequilibrated with 50 mM Tris-HC1 buffer containing 10 mM 2-mercaptoethanol. The enzyme is eluted with the same buffer used for preequilibration of the column at a flow rate of 10 ml/hr. After 5 hr, the flow rate is increased to 20 ml/hr. The initial slow flow rate is to prevent irregularities in migration pattern caused by the high viscosity of the sample. Fractions of 3.3 ml are collected. Proteins are separated primarily in two peaks detected at 260 rim, and CPO activity is eluted with the later peak, Fractions with CPO activity over 40% of peak are combined and ammonium sulfate is added to a concentration of 0.8 M. Phenyl-Sepharose CL-6B Column Chromatography. The fraction obtained in the previous step is loaded on a column of phenyl-Sepharose CL6B (16 x 200 ram) preequilibrated with 0.8 M ammonium sulfate in 20 mM Tris-HC1, pH 7.4, containing 10 mM 2-mercaptoethanol at a flow rate of 15 ml/hr. The column is washed with 30 ml of the same buffer used for preequilibration. The enzyme is eluted with a linear gradient from 0.2 to 0 M ammonium sulfate in 200 ml of the buffer, followed by further washing of the column with 50 ml of the buffer containing 5% (v/v) 2-propanol. The eluate is collected in 2-ml fractions. Coproporphyrinogen oxidase activity is eluted at the end of the gradient or at the boundary to the buffer containing 2-propanol as a single peak that coincides with absorbance at 260 nm. Fractions with the same specific activity as the peak are combined and concentrated by ultrafiltration [Amicon (Danvers, MA) filter membrane YM10; molecular weight cutoff 10,000]. The results of typical purification are summarized in Table I. Purification of Recombinant Mouse Coproporphyrinogen Oxidase from Escherichia coli In the same manner as purified bovine liver CPO, mouse cDNA is isolated 9 and the recombinant cDNA expressed in mammalian cells 9 or in

[41]

COPROPORPHYRINOGEN III OXIDASE FROM BOVINE LIVER

361

TABLE I PURIFICATIONOF COPROPORPHYRINOGEN OXIDASEFROM BOVINE LIVER

Fraction Homogenate Ammonium sulfate fractionation (35-45%) DEAE-Cellulofine Hydroxylapatite Butyl-Toyopearl Cellulofine GCL-2000m Phenyl-Sepharose CL-6B

Protein (mg) 92,000 8,200 630 68 8.7 4.3 0.18

Total activity Specificactivity (× 10-3 units) (units/mgprotein)

Recovery (%)

213 98.1

2.31 11.9

100 46.1

51.2 28.3 19.8 15.8 7.6

81.3 416 2,280 3,670 42,200

24.0 13.3 9.3 7.4 3.6

E. coli 15 to produce a protein with coproporphyrinogen oxidase activity. To elucidate the properties and reaction mechanism of the enzyme, recombinant mouse CPO is purified from E. coli and yields more protein than when purified from mammalian cells.

Construction of Expression Vector of Mouse Coproporphyrinogen Oxidase in pUC18 Mouse full-length CPO is 3.0 kbp and CPO is encoded by 1062 bp, corresponding to 354 amino acid residues. Amino acid terminal analysis of the bovine enzyme suggests that the N terminus of the mature protein starts at residue 32 (serine), corresponding to nucleotide 94.9 The mouse CPO cDNA has restriction sites for SacI at nucleotide 13 and for KpnI at nucleotide 1385. The SacI-KpnI fragment of CPO cDNA is excised from the full-length cDNA and ligated into the multiple cloning site of pUC18 to generate the expression vector pMCO-1. This vector is used to transform E. coli strain JM109. The enzyme expressed in E. coli is composed of 355 residues, 32 amino acid residues longer than the mature enzyme, and the sequence of the N terminus is MITNSSSGAR, in contrast with that of the original cDNA encoding MVPKSSGAR-.

Cultivation of Escherichia coli Carrying Expression Vector Escherichia coli strain JM109 transformed with pMCO-1 is precultured in 10 ml of Luria-Bertani (LB) medium in the presence of sodium ampicillin (100/xg/liter) overnight with shaking at 37 °. The medium is inoculated into 1 liter of LB medium supplemented with sodium ampicillin (100/xg/liter) and 2 mM isopropyl-/3-D-thiogalactopyranoside (IPTG) for 10 hr with shak-

362

HEME

[411

ing at 37°. The cells are collected by centrifugation with a yield of 3-6 g of cells (wet weight) per liter.

Purification of Recombinant Mouse Enzyme from Escherichia coli The procedures for large-scale purification of CPO (50-100 g) have been described previously.IS In this section, a simple small-scale purification procedure using 5-10 g cells is described. Escherichia coli cells (5-10 g wet weight) immediately after culture or from freezer storage are suspended in 30 ml of 0.1 M Tris-HC1 buffer, pH 7.8, containing 50 mM 2-mercaptoethanol. The suspension is sonicated until no intact cells remain, taking care to avoid heating, and then diluted with 50 ml of cold distilled water containing 10 mM 2-mercaptoethanol. The pH value of the solution is adjusted to pH 7.5 and cell debris is removed by centrifugaton at 30,000 g for 30 min. The supernatant is loaded on a column of DEAE-Cellulofine (26 x 400 mm) equilibrated with 20 mM Tris-HC1 buffer, pH 7.6, containing 10 mM 2-mercaptoethanol at a flow rate of 20 ml/hr. The column is washed, at a flow rate of 40 ml/hr, with the buffer used for equilibration of the column until the absorbance monitored at 260 nm returns to the initial level (about 200 ml of buffer). The enzyme is eluted with a linear gradient of NaC1 from 0 to 0.5 M in 800 ml of the buffer at a flow rate of 40 ml/hr and the eluate is collected in 7-ml fractions. The CPO activity is eluted at a position corresponding to 0.1 M NaC1. Fractions with CPO activity over 60% of the peak are combined and the volume of this pooled fraction is less than 5% of the total eluate volume. The pH value of the pooled fraction is adjusted to pH 6.5 with 3 N H3PO4, taking care not to go below pH 6.5. The combined fractions are loaded on a column of hydroxylapatite (16 x 200 mm) preequilibrated with 20 mM potassium phosphate buffer, pH 6.5, containing 10 mM 2-mercaptoethanol at a flow rate of 12 ml/hr. The column is washed with 40 ml of the same buffer used for equilibration of the column. The enzyme is eluted with a linear gradient of 100 ml of the washing buffer to 100 ml of 0.3 M potassium phosphate buffer, pH 7.6, containing 10 mM 2-mercaptoethanol. The activity of CPO is eluted at a position corresponding to 110 mM phosphate, almost coinciding with the peak of absorbance at 260 nm (Fig. 2). Fractions with CPO activity over 60% of the peak value are combined. The enzyme in the pooled fractions is concentrated by ultrafiltration with an Amicon membrane (YM10) or by precipitation with 60% saturation of ammonium sulfate. A typical purification is summarized in Table II. The enzyme purified by this method has a specific activity of more than 90% of that purified by the large-scale method, as

[41]

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Fro. 2. Elution profile of hydroxylapatite column chromatography for purification of recombinant m o u s e C P O from Escherichia coll. Details are described in text. Fraction 1 indicates the starting position of the gradient for elution. The C P O activities in the fractions are shown with a dashed line and absorbance of eluate monitored at 260 n m is shown with a solid line. T h e scales of these parameters are expressed in arbitrary units of the y axis. The concentrations of phosphate in the fractions are shown with a dotted line.

Properties of Bovine and Mouse Coproporphyrinogen Oxidase Molecular Properties of Coproporphyrinogen Oxidase. The molecular size of purified C P O from bovine liver was reported to be 71.6 kDaT; however, that of C P O from yeast was reported to be 35 kDa, 6 as was that from mouse liver. 8 The accurate molecular sizes of CPOs from m a n y sources have b e e n elucidated by molecular cloning. 9-14 Molecular properties such as molecular weight, amino acid composition, and even conformation of the active sites of enzyme are similar in different m a m m a lian species. The molecular size of mouse CPO is 37,255 Da, 9 that of h u m a n is 36,842 Da, l° and even that of yeast is 37,673 Da. 11 The molecular size of bovine C P O is estimated to be 37 kDa. The molecular size of the bovine enzyme has b e e n determined again by sodium dodecyl sulfate

T A B L E II PURIFICATION

OF RECOMBINANT

COPROPORPHYRINOGEN

OXIDASE

FROM

Escherichia coli

Fraction

Protein (mg)

Total activity (× 10 3 units)

Specific activity (units/mg protein)

Recovery (%)

Crude extract DEAE-Cellulofine Hydroxylapatite

375 39.3 8.3

393 221 148

1,050 5,620 17,800

100 56.2 37.7

364

HEME

[41]

(SDS) electrophoresis and gel filtration, in which purified recombinant mouse CPO and other proteins were used as standards and confirmed to be 37 kDa. TM The discrepancy of the reported molecular size of 71.6 and 35 kDa can probably be explained as follows. Purified CPOs from bovine liver and recombinant mouse enzyme are eluted by gel-filtration column chromatography in a sharp peak corresponding to positions of 37 and 41 kDa, respectively. However, both enzymes elute at an earlier position and in a broad peak corresponding to 60 to 70 kDa in the presence of 0.2% (v/v) Tween 20. Both enzymes after purification have a tendency to aggregate gradually to form a polymer if left on ice for 1 week. The purified CPO from bovine liver has no absorption band in the visible region, suggesting that the enzyme has no prosthetic groups. 7 Purified recombinant mouse CPO also shows no characteristic absorption bands of prosthetic groups when highly concentrated. 15 Metal bound to purified CPO from bovine liver was analyzed with inductivity-coupled plasma atomic emission spectrometry with the following results: 0.07 to 0.14 atom of iron/enzyme and 0.06 atom of copper/ enzyme. However, no other metals (Sn, As, Pb, Ni, Hg, Cd, Be, Cr, Co, Se) were detected. 7 In contrast, recombinant mouse CPO contained 1.05 atoms of copper/enzyme and trace amounts of Fe, Cr, and Ni were detected by inductivity coupled plasma atomic emission spectrophotometry (Shimadze ICPS-8000). 15 As described above, it is hard to recognize that the metal bound to CPO is different between bovine and mouse CPOs. Copper is the metal bound to mammalian CPO. Kinetic Properties of Coproporphyrinogen Oxidases. The Km value of the purified CPO from bovine liver is 48/zM 7 and the value of purified mouse recombinant CPO is 47 ~ M . 15 These Km values are different by an order of magnitude from the values of 0.3 and 0.05 /zM obtained from mouse liver CPO 8 and yeast CPO, 6 respectively. The Km values reported by the other groups are 32/xM for yeast, 19 36/zM for tobacco, 2° 0.15/zM for rat liver, 16 and 35 /zM for Chromatium D. 2~ These Km values can be separated into two groups: over 10/xM and less than 1/.~M. This may be caused by the two types of assay method used (i.e., extraction of protoporphyrin from an organic phase with various concentrations of HC1 and the use of radioactivity). In the determination of the Km value it is important to note that CPO activity decreases at higher ionic strength (apparent Ki is T. Yoshinaga and S. Sano, J. BioL Chem. 255, 4727 (1980). 19 R. Poulson and J. W. Polglase, J. Biol. Chem. 249, 6367 (1974). 20 W. P. Hsu and G. W. Miller, Biochem..L 117, 215 (1970). 21 M. Mori and S. Sano, Biochim. Biophys. Acta 264, 252 (1972).

[41]

COPROPORPHYRINOGEN

III OXIDASE FROM

BOVINE LIVER

365

is 0.2 M), which may be brought about by the substrate. The Km value for the crude enzyme is 25 to 30/xM. 7 Purified CPO from bovine liver is activated by various phospholipid or nonionic detergents two- to fourfold, despite inhibitory effects of ionic detergents such as sodium deoxycholate or sodium dodecyl sulfate.7 Purified mouse CPO is also activated by lecithin or neutral detergents. 8 Activation by these compounds is caused by an approximately twofold increase in Vmax and decrease in Km by 40-50%, and this is the reason for the lower Km value of the crude enzyme.7 Coproporphyrinogen oxidase from bovine liver is slightly activated by reducing agents such as dithiothreitol or 2-mercaptoethanol, presumably by protecting the substrate from autoxidation during incubation, because such reducing agents are less effective with shorter incubation periods. Various agents that react with SH groups such as p-chloromercuribenzoic acid, Nethylmaleimide (NEM), or iodoacetate do not inhibit the activity, significantly suggesting no participation of thiol groups in the enzyme reaction. 7 Inhibitory effects of metal chelators on CPO activity were reported 1,2° using crude enzyme. However, no such effects were found with purified bovine liver enzyme] In addition, no effects of metal chelators such as D-penicillamine were seen even with recombinant mouse CPO containing one atom of copper per peptide. Addition of metal ions showed no effect on CPO activity of purified enzyme] As described above, purified CPO has no color at the wavelength where many prosthetic groups show absorption. In contrast, addition of many compounds regarded as prosthetic groups to the assay system of purified bovine CPO resulted had no effect.7 Coproporphyrinogen oxidase from mammalian cells grown under aerobic conditions seems to have no need for prosthetic groups, in contrast to anaerobic cells. Molecular oxygen is believed to be necessary for the catalysis of CPO from aerobic cells. The activity of CPO was not significantly decreased when the reaction mixture was maintained under vacuum with a rotary oil pump without substitution of nitrogen in the reaction tube, suggesting that CPO from aerobic cells has higher affinity for oxygen.22 General Properties of Coproporphyrinogen Oxidase. No prosthetic groups or factors other than copper have been found. The effects of limiting uptake of copper during enzyme synthesis were examined using minimum medium or LB medium in the presence of a copper-chelating agent, and recombinant mouse CPO was synthesized although its activity was reduced. This result strongly supported participation of copper in the enzyme reaction. 15 22 T. Yoshinaga, unpublished data (1996).

366

~EMZ

[41 ]

In many enzymes, copper coordinates with histidine residues. Four histidine residues and one cysteine residue (H148, H158, H197, H227, and C219), conserved in many CPOs, were replaced with alanine residues by site-directed mutagenesis. Strain H158A (H158 to A158) showed complete loss of CPO activity, whereas the amount of protein produced was slightly increased. The enzyme of strain H148A showed decreased stability and low specific activity.15 Coproporphyrinogen oxidase from strain H158A was purified to homogeneity according to the procedure described for normal recombinant mouse CPO, using immunoactivity as an indicator of CPO. The protein of H158A behaved in the same way as the normal enzyme during purification, suggesting that the conformation of the enzyme is conserved after replacement of histidine residue with alanine. No copper atoms were detected in the preparation of H158A. From these results, copper atoms in CPO seem to coordinate with a histidine residue and participate in enzyme catalysis. Chemical modification of bovine CPO also suggested that tyrosine residues play a role in catalysisTM and this should be confirmed by site-directed mutagenesis. Genetic analyses of hereditary coproporphyria indicated that substitution of some amino residues caused decreased CPO activity.23'24 Studies to elucidate the reaction mechanism of the enzyme began more than 20 years ago, using substrate analogs because of the difficulty in obtaining the enzymeY '26 fl-Hydroxypropionate at positions 2 and 4 of porphyrin is thought to be one of the intermediates of the reactionJ s,z7,28 The question has been raised as to why CPO selectively catalyzes only two propionyl groups of four at positions 2 and 4. Elder et al. proposed a mechanism in which porphyrins containing a sequence of substituents of methyl-methyl-propionyl-methyl or vinyl-methyl-propionyl-methyl have higher affinity for the enzyme and the propionyl group is converted to a vinyl group. 3 According to this proposal, the substrate will rotate 90° after conversion of the first propionyl group at position 2 to a vinyl group? This was also confirmed using porphyrins with a/3-hydroxypropionyl group at position 2, position 4, or both. TM

23 p. Martasek, Y. Nordmann, and B. Grandchamp, Hum. Mol. Genet. 3, 477 (1994). 24 H. Fujita, H. Kohno, S. Taketani, N. Nomura, K. Furuyama, R. Akagi, T. Terajima, R. A. Galbrain, and S. Sassa, Hum. Mol. Genet. 3, 1807 (1994). 25 A. R. Battersby and E. McDonald, in "Porphyrins and Metalloporphyrins" (K. M. Smith, ed.), p. 61. Elsevier, New York, 1975. 26 A. R. Battersby, E. Hunt, E. Edword, and J. Moron, J. Chem. Soc. Perkin I, 2917 (1972). 27 S. Sano, J. BioL Chem. 241, 5276 (1966). z8 M. Akhtar, in "Biosynthesis of Tetrapyrroles" (P. M. Jordan, ed.), p. 67. Elsevier, New York, 1991.

[42]

COPROPORPHYRINOGEN

III OXIDASE FROM YEAST

367

Some points remain unclear concerning the subunit structure of CPO (i.e., (1) whether the molecular size of CPO is 71 or 35 kDa, and (2) whether CPO has a monomeric or a homodimeric structure). The molecular size of the peptide was determined to be 37 kDa by genetic analysis. As described above, purified bovine liver enzyme and the recombinant enzyme behave as monomeric structures during gel-filtration column chromatography. It is possible that CPO is a dimeric enzyme that forms an active site between the subunits, but which in the absence of substrate dissociates to monomers. It is difficult to determine the molecular size in the presence of substrate by gel-filtration column chromatography, because the substrate is not sufficiently stable during chromatography. To examine this hypothesis, recombinant mouse CPO activity was measured in the presence of various molar ratios of inactive point-mutated recombinant mouse CPO (H158A). Because H158A-CPO conserves the conformation of the native enzyme, active CPO and inactive H158A-COP should form heterodimers with an incomplete active site between the subunits and the total enzyme activity would consequently decrease. However, the enzyme activity was not decreased by the addition of the inactive CPO. 22 The hypothesis is also unlikely with reference to the results concerning the rotation mechanism of substrate described above. 3'18 Coproporphyrinogen oxidase is therefore likely to be a monomeric enzyme in mammalian cells.

[42] P u r i f i c a t i o n a n d P r o p e r t i e s o f C o p r o p o r p h y r i n o g e n III O x i d a s e f r o m Y e a s t

By P I E R R E

LABBE

Introduction Coproporphyrinogen III oxidase (CPO) is an enzyme of the heme and chlorophyll biosynthetic pathways that catalyzes the oxidative decarboxylation of the 2- and 4-propionyl groups of coproporphyrinogen III (coprogen) into vinyl groups to form protoporphyrinogen IX (protogen). x'2

t S. S a n o a n d S. G r a n i c k , J. Biol. Chem. 236, 1173 (1961). 2 R . J. P o r r a a n d J. E. F a l k , Biochem. J. 90, 69 (1964).

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