119
Biochimica et Biophysica Acta, 1040 (1990) 119-129
Elsevier BBAPRO 33701
Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase Judith Frydman,
M a r i a L. T o m a r o , J o r g e R o s e n f e l d a n d R o s a l i a B. F r y d m a n
Facultad de Farmacia y Bioqulrnica Unioersidad de Buenos Aires, Buenos Aires (Argentina)
(Received 10 November 1989)
Key words: Biliverdin; Thiol residue; Molecular form; NADPH binding; Dehydrogenase; (Rat liver)
Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P) + oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide ( N E M ) / m o l enzyme's S H = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of N E M / e n z y m e ' s S H = 300, two thiol residues were aikylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by N E M or by iodoacetamide. Biliverdin as well as the co-substrate N A D P H protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group hinds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxai phosphate the catalytic activity was irreversibly abolished. The location of a lysiue residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thioi and ¢-NH 2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the N A D P H binding site was inferred from the shifts in the UVm~x of N A D P H from 340 um to 327 nm and of 3-acetyl N A D P H from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by N A D P H but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.
Introduction Heme is degraded in mammals by an ubiquitous microsomal heme oxygenase to give biliverdin IX a, which is then reduced by a cytosolic biliverdin reductase to give bilirubin IX a [1]. Biliverdin reductase (EC 1.3.1.24, bilirubin :NAD(P) + oxidoreductase) was first isolated and partially purified from rat liver [2] and later from other sources [3-6]. The enzyme has been
Abreviations: NEM, N-ethylmaleimide; NPhM, N-phenylmaleimide; DTNB, 5,5'-dithiobis(2-nitrobenzoate); TCPK, L-l-tosylamide-2phenylethylchloromethylketone; BvR, bifiverdin reductase; FPLC, fast-performance liquid chromatography; PMSF, phenylmethylsulfonylfluoride; MF1 and MF3, molecular form 1 and 3, respectively. Correspondence: R.B. Frydman, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Jurtin 956, Buenos Aires, Argentina.
purified and several of its properties have been studied [3,4,7-10]. However, its primary structure is still unknown and crystallographic data are not available. Biliverdin reductase is an NADPH-dependent enzyme which reduces the C-10-C-11 double bond of 5,10,15-bilatrienes to give 5,15-biladienes (bilirubins). Its substrate specificity has been extensively studied [8,9] and it has been shown that substrate activity requires that the bilitrienes should be substituted with at least two propionate side chains which might be located at any of the eight fl positions of the pyrrole rings [9]. The substitution of one of the two propionate side chains by an acetate residue results in a bilitriene devoid of substrate activity [9]. The substrate specificity of the enzyme is therefore a rather broad one; the reductase was found to efficiently reduce a large number of synthetic bilitrienes even when they were substituted with eight carboxylate residues (bactobilins). N A D P H could be
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
120 replaced by N A D H , 3-acetyl N A D P H and deamino N A D P H with retention of good substrate activity for the reduction of the natural type IX biliverdins as well as for the reduction of the synthetic biliverdins of type I and the harderobiliverdins [8]. It was found that the rat liver enzyme was present in two molecular forms; a major molecular form 1 (MF1) with M r = 34000 and a minor molecular form 2 with M r = 56 000 [11,12]. Biliverdin reductase from ox kidney was also found to exist in two molecular forms which might be similar to those mentioned above [7]. Under conditions of enhanced hemoprotein degradation, an NAD÷-dependent peroxisomal dehydrogenase is induced which transforms MF1 into a new molecular form 3 (MF3) [13]. This conversion results from the oxidation of one of the three thiol residues present in MF1 to form a disulfide linkage in MF3 ( M r = 68000), which is therefore a dimer of MF1. Biliverdin reductase from rat kidney and spleen consists of only one molecular form which is identical in many of its properties to MF1 found in the liver. The kidney and spleen enzymes were not oxidized by the peroxisomal dehydrogenase to give MF3, very likely due to small significant molecular differences within the MF1 of rat liver [10]. It is known that biliverdin reductase contains three thiol residues [4,7,10]. Alkylation studies using 4-vinylpyridine and 4-dimethylamino-azobenzene-4'-iodoacetamide showed that in molecular form 1 the three SH residues are located in a single peptide isolated by CNBr-fragmentation [10]. It is known from biliverdin chemistry that the C-10 methine carbon is a strong electrophile and that electron rich donors (e.g., thiol residues) add readily to that carbon to form a thioalkyl derivative [14]. Cysteine adds to biliverdin in fractions of seconds in a polar but aprotic solvent such as dimethylsulfoxide (DMSO); the adduct is rapidly decomposed in the presence of water. It is therefore no surprise that cysteine residues were shown to be essential for the activity of biliverdin reductase. The latter was found to be inactivated by alkylating agents and by sulfhydryl reagents [3,4,7,11]. The aim of the present study is to identify how many of the three thiol residues of MF1 are involved in the enzymatic activity; how many of them bind to the substrate, and whether it is possible to differentiate the thiol involved in substrate binding from that involved in the conversion of MF1 into MF3. In addition, evidence on the nature of the co-substrate binding to the enzyme were also looked for in order to establish constraints for future crystallographic studies.
Experimental procedures Materials Biliverdins IX a and IX fl were prepared by the chemical oxidation of heroin IX and separated as described elsewhere [15]. N A D P H , N A D +, N A D H , N-
ethylmaleimide, N-phenylmaleimide, pyridoxal phosphate, butanedione, dithiothreitol, PMSF, o-phthalaldehyde, DEAE-cellulose, CM-cellulose, 5,5'-dithiobis(2-nitrobenzoate) and p-hydroxymercuriphenylsulfonate were from Sigma Sephadex G-25, G-100 and Agarose-hexanoyl-NADP ÷ affinity gel were from Pharmacia. All the other chemicals used were of the highest purity available. The solvents were from Merck (Darmstadt). Trypsin ( T C P K treated) and thermolysin were from Worthington.
Methods Isolation and purification of the molecular forms of biliverdin reductase. The molecular forms of rat liver biliverdin reductase were obtained from normal and CoC12-treated Wistar rats (180-200 g) and separated as described elsewhere. They were then purified to homogeneity by affinity chromatography and FPLC essentially as described for molecular form 1 [10]. Kidney biliverdin reductase was prepared from Wistar rats (180-200 g) and purified to homogeneity as described elsewhere [10]. Preparation of the converting enzyme. The converting enzyme was prepared from the livers of Wistar rats (180-200 g) which were treated with CoC12 for 2.25 h. Peroxisomes were obtained and the enzyme was solubilized with 0.5% DOCA as described elsewhere [13]. The solubilized enzyme was then ultracentrifuged at 150 000 × g. The enzyme was further purified by a sequential use of DEAE-ceUUlose, CM-cellulose, Sephacryl S-200 and finally by FPLC (the details of this purification will be described elsewhere). The resulting enzyme had a requirement for N A D ÷ and was 90% pure as judged from polyacrylamide gel electrophoresis under non-denaturing conditions using silver nitrate to stain the proteins. Assay procedures. Biliverdin reductase activity was assayed at 37°C for 10 min. The incubation mixture contained in a final volume of 100 /~1:10 mM of potassium phosphate buffer (pH 7.4), 500 /LM of N A D P H , 13/xM of biliverdin IX a or biliverdin IX /3 and enzyme (200 ng, specific activity 3500 units/rag protein) unless otherwise indicated. Bilirubin formation was measured at 455 nm (c = 50 m M -1 • cm-1). The conversion of MF1 into MF3 was measured by preincubation of MF1 with the converting enzyme. The reduction rate of biliverdin IX ct by MF1 is twice as fast as the reduction rate of biliverdin IX fl by the same form, while MF3 reduces both biliverdin isomers at the same rate. The transformation of MF1 to MF3 by the converting enzyme could therefore be followed by measuring the relative reduction rate of both isomers. The incubation mixtures contained in a final volume of 100 /~1:10 /~mol of potassium phosphate buffer (pH 7.4), MF1 (200 ng), N A D ÷ (100 nmol) and converting en-
121 zyme (100 ng). The mixture was preincubated for 10 min at 3 7 ° C after which N A D P H (500/~M) and either biliverdin IX a or IX fl (13 #M) were added and the mixture was incubated in a final volume of 200 #1. BvR activity was measured as described above. The increase of the reduction rate of biliverdin IX fl over that of a control (in the absence of either converting enzyme or N A D ÷) measured the presence of MF3. Analytical procedures. Protein concentrations were determined by the method of Bradford[16] using serum albumin as standard. Absorption and fluorescence spectra were recorded at room temperature (18-22°C) on a Hitachi U-2000 double-beam spectrophotometer and on an Aminco Bowman fluorometer, respectively. Polyacrylamide gel electrophoresis. Denaturing SDSpolyacrylamide electrophoresis was essentially performed according to the method of Laemmli [17] using a 3% stacking gel (pH 6.8). The samples were electrophoresed in 11% polyacrylamide resolving gel (pH 8.8) using Bromophenol blue as a tracking dye. The running buffer was Tris-glycine (pH 8.3). The protein samples were routinely prepared by mixing them with the cracking buffer made up of 120 mM Tris-HC1 buffer (pH 6.8), 2% SDS, 20% glycerol and, when indicated, 10% 2-mercaptoethanol. They were heated for 10 min at 100°C. Native gel electrophoresis was performed on 7.5% polyacrylamide gels using the neutral discontinuous buffer system of Williams and Reisfeld [18]. The runs were performed using Tris-barbital buffer (pH 7.5). The electrophoresis was carried out for 30 min at 35 V and then for 4 h at 250 V with Bromophenol blue as a tracking dye. Routinely, 75 ng of pure enzyme was run for silver staining of gels and 185 ng for Western blotting. The gel protein staining was performed either with Coomasie brilliant blue R-250 (0.25% in 50% methanol, 10% acetic acid) or with silver nitrate as described Ref. 10. Chemical modifications of biliverdin reductase. Purified biliverdin reductase (7/~g protein, a single band on native and SDS-PAGE when stained with silver nitrate) was preincubated at 37°C with o-phthalaldehyde in 50 m M Hepes-NaOH (pH 8.0) in the presence of 1% ethanol in a final vol. of 100 #1. Preincubations were performed either at different time periods using 10 mM o-phthalaldehyde, or during a 15 min time period using different o-phthalaldehyde concentrations. At the end of the treatments BvR activity was determined as described above by completing the incubation mixture with biliverdin IX a and N A D P H . When fluorescence was measured, the enzyme preincubated with o-phthalaldehyde was diluted to a final vol. of 0.4 ml and the fluorescence measurements were made with an Aminco Bowman spectrofluorometer. The excitation and emission wavelengths were 337 and 408 nm, respectively.
Succinylation of the enzyme was performed by addition of succinic anhydride (1000 molar excess) to 0.2 nmol (7/~g) of the enzyme in 0.2 ml of phosphate buffer (pH 7.4). The p H of the solution was maintained by addition of M potassium hydroxide. After 30 min the treated enzyme was passed through a Sephadex G-25 column equilibrated with 100 m M phosphate buffer (pH 7.5). When the succinylation was carried out in the presence of either biliverdin or N A D P H , the enzyme was first preincubated with the former for 15 min at 0 ° C and then succinylated as described above. When hydroxylamine was used to probe if succinylation acylated an c-NH 2 of lysine, the former was added to the succinylated enzyme at a 0.1 M final concentration (pH 9.0) and the mixture was incubated at 0 - 4 ° C for 2 h before gel filtration to cleave any O- or S-succinyl derivatives [19]. Modification of the enzyme with 2,3butanedione was performed by preincubating the former (7/~g) in 50 mM borate buffer (pH 8.5) at 37 ° C in the dark, in the presence or absence of the dione. Aliquots of the control and treated enzyme mixtures were then added to the BvR assay mixture at a final 5-fold dilution. When the reaction of butanedione was examined on the enzyme which had been previously preincubated at 0 ° C for 15 rain with either biliverdin, N A D P H or both of them, the reagent was added and then incubated for 10 min at 37°C. Controls were run by using the same procedure but in the absence of butanedione. Determination of the reactive thiol groups. The number of reactive thiol groups of BvR was measured by titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as described by DeLuca and McElroy [20]. The increase in absorbance at 412 nm of a reaction mixture (1.0 ml) containing 7/~g of purified BvR in 0.1 M potassium phosphate buffer (pH 7.4) and 500 # M DTNB, was determined at 25°C against a reference reaction mixture which contained D T N B in buffer. The thiol content was calculated assuming c = 14.1 m M -~- cm -1 at 412 nm for the reaction product. The reaction was complete after 30 min. For determinations performed under denaturing conditions, the enzyme was previously pretreated with 6 M guanidinium hydrochloride. Results
Evidence that different thiol groups are involved in the catalytic activity and in the dimerization of molecular form 1 in liver biliverdin reductase Treatment of MF1 of rat liver biliverdin reductase with p-chloromercurisulphonate, iodoacetamide, N E M and N P h M at various concentrations showed that the inhibition of the conversion of MF1 into MF3 was achieved at lower concentrations and shorter incubation times than the inhibition of the enzymatic activity (Fig. 1). p-Chloromercuribenzoate inhibited 90% of the conversion at concentrations where only 15% of the en-
122
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Fig. 4. Loss of bgiverdm reductase activity by treatment with o-phtha]aldehyde. (A) As a function of the reagent concentration, preincubations
were performed for 15 rain at 37°C. (B) Time-course of the loss of the reductase activity (o), and increase in fluorescence intensity (e) of biliverdin reductase, MFI. The enzyme was treated with 10 mM o-phthalaidehyde for the indicated times. Parallel experiments for measuring activity and fluorescence were run. The data were presented as Ft - Fo vs. time, where Ft represents the fluorescence at the time t and F0 at time to. The excitation and emission wavelengthswere 337 nm and 408 nm, respectively.
126 cence (48%). It is obvious that at a given concentration of o-phthalaldehyde the loss of enzymatic activity parallels a corresponding increase in the relative fluorescence.
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Inactivation of biliverdin reductase by butanedione and protection by substrates It is known that guanidinium groups of arginine can function as contact sites for the anionic phosphate groups of the pyridine nucleotides [22]. The same basic group could also stabili7e, by electrostatic binding, the carboxylate anions of the propionate side chains of biliverdin. It followed that the reaction of the enzyme with butanedione could prove the possible involvement of arginine residues in its activity. As can be seen in Fig. 5, butanedione inactivated the enzyme both in a concentration- and in a time-dependent manner. The inhibition by butanedione was completely countered by preincubation of the reductase with N A D P H , while biliverdin did not affect the inactivation of the enzyme by 10 or 15 m M butanedione (data not shown). Therefore, at least one arginine residue is in the close proximity of the N A D P H binding site, but removed enough from the biliverdin binding site. This arginine residue could therefore be the stabilizing cation of the phosphate residues which are nearer to the adenine moiety of the nucleotide.
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Spectroscopic evidence for the presence of a positively charged ion at the binding site of NADPH It is known that the UVm~ of N A D P H at 340 nm shifts to 325 nm when the latter is complexed with horse liver alcohol dehydrogenase, bovine heart lactate dehydrogenase or rat liver lactate dehydrogenase [23]. A similar spectroscopic shift was observed when 3-acetyl
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Fig. 6. Spectral shifts of NADPH and 3-acetyl NADPH on bindin8 to biliverdin reductase. (A) Spectra of ( - - - - - ) NADPH when added t o bovine serum albumin (1 m s / m l ) in 20 mM phmphate buffer (pH 7.4) and ( ) NADPH added to bih'vexdin redu~tase (160 ttg/ml) in the same buffer. (B) Spectra of ( - - - - - - ) 3-acetyl-NADPH and ( ) 3-acetyi-NADPH plus b i l i v e r d i n - ~ . The 3-acetyl-NADPH w a s obtained from 3-acetyl-NADP+ by chemical reduction with dithionite. Inset of (B) shows the difference spectrum between ( ) and ( - - - - - - ) of the last spectra.
127 about 3/~ from the dihydropyridine ring would increase the observed transition energy for light absorption by the ring and account for the aforementioned shifts. The spectra of the NADPH complex showed a UVm~ at 327 nm, while NADPH itself as well as NADPH in the presence of BSA showed the expected maximum at 340 nm (Fig. 6A). The 3-acetyl NADPH-MF1 complex showed a UVm=, at 348 pm while 3-acetyl NADPH itself or in the presence of BSA gave the expected UVmx at 360 nm (Fig. 6B). The differential spectra at two different nucleotide concentrations can be seen in the inset to Fig. 6B. We have already shown that 3-acetyl NADPH is a good co-substrate for biliverdin reductase [8]. These spectral data provided evidence for the presence of a positive charge in the vicinity of the binding site of NADPH in the reductase similar to those present in the dehydrogenases. Crystallographic data have shown the presence of arginine guanidinium cations in the active sites of lactate dehydrogenase as well as of glyceraldehyde-3-phosphate dehydrogenase [25], while in horse liver alcohol dehydrogenase the positive charge is a.pparently related to the Zn2÷ atom located about 3-4 A from the nicotinamide ring [26]. In the case of biliverdin reductase, the positive charge very
likely resides on the nitrogen of the alkylammonium ion formed by the E-amino group of the lysine residue which was shown to be closed to the substrate binding thiol residue (see above). In NADH-cytochrome b5 reductase, a cysteine [27] and a lysine residue [19] were shown to be involved in NADH binding.
Inactivation of bilioerdin reductase by trypsin and protection by substrates The presence of lysine and arginine residues at or near the active center of the reductase called for an examination of the effect of trypsin on the activity of molecular form 1 of the enzyme. A 2 mM trypsin concentration inactivated 70% of the enzyme at 5 min and 90% of the enzyme after 11 rain (Fig. 7). Both the substrate and the co-substrate strongly protected the enzymatic activity and in the presence of each of them 80% of the activity was preserved after a 10 rain preincubation with trypsin (Fig. 7). These results could be expected from an enzyme with a lysine residue at the active site and with an arginine residue needed for the stabilization of the co-substrate. The protection by either substrate or co-substrate suggest that the fast attack by trypsin takes place at the lysine (or arginine) residues at the active center and this is supported by the fact that the degraded reductase did not shift the pyridine nucleotide spectra any further. Discussion
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TIME, rain Fig. 7. Time dependent inactivation of biliverdin reductase by trypsin. MF1 (2/~M) was treated with trypsin (2 mM) in the absence (O) and presence of either (,,) biliverdin (11 btM) or (zx) N A D P H (500/*M). The enzyme was preincubated 10 rain at 370C with each of the substrates before trypsin was added. After the preincubation with trypsin during the indicated times, soybean trypsin inhbitor (4 raM) was added and the activity was assayed as described in Experimental procedures.
An analysis of the structural features of molecular form 1 of biliverdin reductase will be helpful to understand its dynamics when the primary sequence and crystallographic data will outline its architecture. The reduction of biliverdin to bilirubin by a hydride transfer from a reduced nicotinamide ring bears similarities to the reduction of carbonyl groups by the dehydrogenases. It has been repeatedly suggested that in the latter a sp2-sp 3 transition takes place during catalysis [221. The reversible addition of thioles to the C-10 methine of biliverdin and to carbonyl groups have similar thermodynamic and kinetic parameters for the equilibrium reaction [14]. It is therefore conceivable that there is a convergence of the active center geometries of some dehydrogenases and of biliverdin reductase. It is known that in glyceraldehyde-3-phosphate dehydrogenase a thiol residue is involved in the hydride transfer to the carbonyl group, very likely through a prior addition of cysteine to form a tetrahedral intermediate [22]. We have shown that a thiol residue present at the active center binds to biliverdin (Table I). Hydride transfer to the C-S bond will result in the fast cleavage of the adduct with formation of bilirubin. The finding that a lysine residue is located at about 3 ,~ from the thiol group at the active center (Fig. 4 and Table II) and the
128 Pyridoxal phosphate and biliverdin also conferred full protection to the enzyme from alkylating agents, very likely due to the presence of a lysine residue at the active center. This is supported by the formation of an isoindol derivative in the presence of o-phthalaldehyde which could only result from the reaction of neighboring thiol and c-NH 2 residues with the reagent. The cationic ion formed from the c-NH 2 of the lysine residue could be responsible for the hypsochromic shift observed in the UVm~, of the reduced nucleotide (NADPH and 3-acetyl-NADPH) when they bind to the enzyme. The protection of the enzymatic activity in the presence of NADPH from chemical modification by butanedione suggest that arginine residues are involved in salt-like linkages with phosphate residues of the co-substrate.
/ Fig. 8. A model of the ternary transition-state complex of biliverdin reductase MF1.
possibility that its c-NH 2 is present in the form of an ammonium ion at a 3-4 A distance from the nicotinamide ring allows us to outline a picture for the transition state of the enzyme-co-enzyme substrate ternary complex (Fig. 8). The evidence that an arginine residue is also essential for enzymatic activity and is placed at the binding site of NADPH could also be the origin of the UV~, shift observed for the pyridine nucleotides after binding to the enzyme (Fig. 6). In the absence of crystallographic data it is difficult to decide on this issue, since either the lysine or arginine residues could be attached to flexible loops which undergo conformational changes during catalysis. These residues could move into the active center during the formation of the ternary complex (see Ref. 25). We favor the location of the lysine residue near the binding site of biliverdin and NADPH rather than the arginine residue since biliverdin does not prevent the inactivation of the enzyme by butanedione.
Conclusion Of the three thiol residues present in MF1 of biliverdin reductase only two could be modified with alkylating agents under nondenaturing conditions. The more exposed thiol group is that involved in the conversion of MF1 into MF3 of the liver reductase. The thiol group involved at the active site in the binding of biliverdin is less reactive. This binding was not affected by guanidinium chloride and results very likely from the formation of a C-S bond. Full protection of the enzyme from alkylating agents could only be achieved by the simultaneous presence of both biliverdin and NADPH.
Acknowledgements This work was made possible by grants (GM-11973) from the National Institutes of Health and from CONICET (Argentina).
References 1 Frydman, R.B. and Frydman, B. (1987) Accounts Chem. Res. 20, 250-256. 2 Tenhunen, R., Ross, M.E., Marver, H.S. and Schmid, R. (1970) Biochemistry 9, 298-303. 3 Noguchi, M., Yoshida, T. and Kikuchi, G. (1979) J. Biochem. 86, 833-848. 4 Kutty, R.K. and Maines, M.D. (1981) J. Biol. Chem. 256, 39563962. 5 Yoshinaga, T., Sassa, Sh. and Kappas, A. (1982) J. Biol. Chem. 257, 7786-7793. 6 Fang, L.Sh. and Lai, Ch.Ch. (1987) Comp. Biochem. Physiol. 88B, 1151-1155. 7 Rigney, E.M., Phillips, O. and Mantle, T.J. (1988) Biochem. L 255, 431-435. 8 Tomato, M.L., Frydman, R.B., Awrnch, J., Valasinas, A., Frydman, B., Pandey, R.K. and Smith, K.M. (1984) Biochim. Biophys. Acta 791, 350-356. See Bonnett, R. (1975) in Metabolism and Chemistry of Bilirnbin and Related Tetrapyrrois (Bakkcn, A.F. and Fong, J., eds.), p. 212. 9 Frydman, R.B., Tomaro, M.L., Rosenfeld, J., Awrnch, J., Sambrotta, L., Valasinas, A. and Frydrnan, B. (1987) Biochim. Biophys. Acta 916, 500-511. 10 Cascone, O., Frydman, R.B., Ferrara, P., Tomaro, M.L. and Rosenfeld, J. (1989) Eur. J. Biochem. 179, 123-130. 11 Frydman, R.B., Tomato, M.L., Awruch, J. and Frydman, B. (1982) Biochim. Biophys. Res. Commun. 165, 752-758. 12 Frydman, R.B., Tommy, M.L., Awruch, J. and Frydman, B. (1983) Biochim. Biophys. Aeta 759, 257-263. 13 Frydman, R.B., Tomato, M.L., Awrnch, J. and Frydman, B. (1984) Biochim. Biophys.Res. Commua. 121,249-254. 14 Falk, H., Muller, N. and Sehlederer, Th. (1980) Mh. Chem. 111, 159-175 (and refereaaees therein). 15 Frydman, R.B., Awrueh, J., Tomaro, M.L. and Frydman, B. (1979) Biochim. Biophys. Res. Commun. 87, 928-935. 16 Bradford, M. (1976) Anal. Biochem. 72, 248-254. 17 Laemmli, U.K. (1970) Nature 227, 680-685.
129 18 Williams, P.E. and Reisfeld, R.A. (1964) Ann. N.Y. Acad. Sci. 121, 373-381. 19 Loverde, A. and Strittmatter, P. (1968) J. Biol. Chem. 243, 57795787. 20 De Luca, M. and McElroy, W.D. (1966) Arch. Biochem. Biophys. 116, 103-107. 21 Purl, R.N., Bhatnagar, D. and Roskoski, Jr. R. (1985) Biochemistry 24, 6499-6508. 22 Jeffrey, J. (1982) in Stereochemistry (Tamm, Ch., ed.), pp. 113-160 (and references therein), Elsevier, Amsterdam.
23 Van Eys, J., Stolzenbach, F.E., Sherwood, L. and Kaplan, N.O. (1958) Biochirn. Biophys. Acta, 27, 63-83. 24 Kosower, E. (1962) Molecular Biochemistry, pp. 213-219, McGraw Hill Series in Advanced Chemistry. 25 Garavito, R.M., Rossmann, M.G., Argos, P. and Eventoff, W. (1977) Biochemistry 16, 5065-5071. 26 Eklund, H., Samarma, J.P. and Jones, T.A. (1984) Biochemistry 23, 5982-5996. 27 Hackett, C.S., Novoa, W.B., Ozols, J. and Strittmatter, Ph. (1986) J. Biol. Chem. 261, 9854-9857.
Biochimica et Biophysica Acta, 1040 (1990) 119-129
Elsevier BBAPRO 33701
Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase Judith Frydman,
M a r i a L. T o m a r o , J o r g e R o s e n f e l d a n d R o s a l i a B. F r y d m a n
Facultad de Farmacia y Bioqulrnica Unioersidad de Buenos Aires, Buenos Aires (Argentina)
(Received 10 November 1989)
Key words: Biliverdin; Thiol residue; Molecular form; NADPH binding; Dehydrogenase; (Rat liver)
Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P) + oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide ( N E M ) / m o l enzyme's S H = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of N E M / e n z y m e ' s S H = 300, two thiol residues were aikylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by N E M or by iodoacetamide. Biliverdin as well as the co-substrate N A D P H protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group hinds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxai phosphate the catalytic activity was irreversibly abolished. The location of a lysiue residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thioi and ¢-NH 2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the N A D P H binding site was inferred from the shifts in the UVm~x of N A D P H from 340 um to 327 nm and of 3-acetyl N A D P H from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by N A D P H but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.
Introduction Heme is degraded in mammals by an ubiquitous microsomal heme oxygenase to give biliverdin IX a, which is then reduced by a cytosolic biliverdin reductase to give bilirubin IX a [1]. Biliverdin reductase (EC 1.3.1.24, bilirubin :NAD(P) + oxidoreductase) was first isolated and partially purified from rat liver [2] and later from other sources [3-6]. The enzyme has been
Abreviations: NEM, N-ethylmaleimide; NPhM, N-phenylmaleimide; DTNB, 5,5'-dithiobis(2-nitrobenzoate); TCPK, L-l-tosylamide-2phenylethylchloromethylketone; BvR, bifiverdin reductase; FPLC, fast-performance liquid chromatography; PMSF, phenylmethylsulfonylfluoride; MF1 and MF3, molecular form 1 and 3, respectively. Correspondence: R.B. Frydman, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Jurtin 956, Buenos Aires, Argentina.
purified and several of its properties have been studied [3,4,7-10]. However, its primary structure is still unknown and crystallographic data are not available. Biliverdin reductase is an NADPH-dependent enzyme which reduces the C-10-C-11 double bond of 5,10,15-bilatrienes to give 5,15-biladienes (bilirubins). Its substrate specificity has been extensively studied [8,9] and it has been shown that substrate activity requires that the bilitrienes should be substituted with at least two propionate side chains which might be located at any of the eight fl positions of the pyrrole rings [9]. The substitution of one of the two propionate side chains by an acetate residue results in a bilitriene devoid of substrate activity [9]. The substrate specificity of the enzyme is therefore a rather broad one; the reductase was found to efficiently reduce a large number of synthetic bilitrienes even when they were substituted with eight carboxylate residues (bactobilins). N A D P H could be
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120 replaced by N A D H , 3-acetyl N A D P H and deamino N A D P H with retention of good substrate activity for the reduction of the natural type IX biliverdins as well as for the reduction of the synthetic biliverdins of type I and the harderobiliverdins [8]. It was found that the rat liver enzyme was present in two molecular forms; a major molecular form 1 (MF1) with M r = 34000 and a minor molecular form 2 with M r = 56 000 [11,12]. Biliverdin reductase from ox kidney was also found to exist in two molecular forms which might be similar to those mentioned above [7]. Under conditions of enhanced hemoprotein degradation, an NAD÷-dependent peroxisomal dehydrogenase is induced which transforms MF1 into a new molecular form 3 (MF3) [13]. This conversion results from the oxidation of one of the three thiol residues present in MF1 to form a disulfide linkage in MF3 ( M r = 68000), which is therefore a dimer of MF1. Biliverdin reductase from rat kidney and spleen consists of only one molecular form which is identical in many of its properties to MF1 found in the liver. The kidney and spleen enzymes were not oxidized by the peroxisomal dehydrogenase to give MF3, very likely due to small significant molecular differences within the MF1 of rat liver [10]. It is known that biliverdin reductase contains three thiol residues [4,7,10]. Alkylation studies using 4-vinylpyridine and 4-dimethylamino-azobenzene-4'-iodoacetamide showed that in molecular form 1 the three SH residues are located in a single peptide isolated by CNBr-fragmentation [10]. It is known from biliverdin chemistry that the C-10 methine carbon is a strong electrophile and that electron rich donors (e.g., thiol residues) add readily to that carbon to form a thioalkyl derivative [14]. Cysteine adds to biliverdin in fractions of seconds in a polar but aprotic solvent such as dimethylsulfoxide (DMSO); the adduct is rapidly decomposed in the presence of water. It is therefore no surprise that cysteine residues were shown to be essential for the activity of biliverdin reductase. The latter was found to be inactivated by alkylating agents and by sulfhydryl reagents [3,4,7,11]. The aim of the present study is to identify how many of the three thiol residues of MF1 are involved in the enzymatic activity; how many of them bind to the substrate, and whether it is possible to differentiate the thiol involved in substrate binding from that involved in the conversion of MF1 into MF3. In addition, evidence on the nature of the co-substrate binding to the enzyme were also looked for in order to establish constraints for future crystallographic studies.
Experimental procedures Materials Biliverdins IX a and IX fl were prepared by the chemical oxidation of heroin IX and separated as described elsewhere [15]. N A D P H , N A D +, N A D H , N-
ethylmaleimide, N-phenylmaleimide, pyridoxal phosphate, butanedione, dithiothreitol, PMSF, o-phthalaldehyde, DEAE-cellulose, CM-cellulose, 5,5'-dithiobis(2-nitrobenzoate) and p-hydroxymercuriphenylsulfonate were from Sigma Sephadex G-25, G-100 and Agarose-hexanoyl-NADP ÷ affinity gel were from Pharmacia. All the other chemicals used were of the highest purity available. The solvents were from Merck (Darmstadt). Trypsin ( T C P K treated) and thermolysin were from Worthington.
Methods Isolation and purification of the molecular forms of biliverdin reductase. The molecular forms of rat liver biliverdin reductase were obtained from normal and CoC12-treated Wistar rats (180-200 g) and separated as described elsewhere. They were then purified to homogeneity by affinity chromatography and FPLC essentially as described for molecular form 1 [10]. Kidney biliverdin reductase was prepared from Wistar rats (180-200 g) and purified to homogeneity as described elsewhere [10]. Preparation of the converting enzyme. The converting enzyme was prepared from the livers of Wistar rats (180-200 g) which were treated with CoC12 for 2.25 h. Peroxisomes were obtained and the enzyme was solubilized with 0.5% DOCA as described elsewhere [13]. The solubilized enzyme was then ultracentrifuged at 150 000 × g. The enzyme was further purified by a sequential use of DEAE-ceUUlose, CM-cellulose, Sephacryl S-200 and finally by FPLC (the details of this purification will be described elsewhere). The resulting enzyme had a requirement for N A D ÷ and was 90% pure as judged from polyacrylamide gel electrophoresis under non-denaturing conditions using silver nitrate to stain the proteins. Assay procedures. Biliverdin reductase activity was assayed at 37°C for 10 min. The incubation mixture contained in a final volume of 100 /~1:10 mM of potassium phosphate buffer (pH 7.4), 500 /LM of N A D P H , 13/xM of biliverdin IX a or biliverdin IX /3 and enzyme (200 ng, specific activity 3500 units/rag protein) unless otherwise indicated. Bilirubin formation was measured at 455 nm (c = 50 m M -1 • cm-1). The conversion of MF1 into MF3 was measured by preincubation of MF1 with the converting enzyme. The reduction rate of biliverdin IX ct by MF1 is twice as fast as the reduction rate of biliverdin IX fl by the same form, while MF3 reduces both biliverdin isomers at the same rate. The transformation of MF1 to MF3 by the converting enzyme could therefore be followed by measuring the relative reduction rate of both isomers. The incubation mixtures contained in a final volume of 100 /~1:10 /~mol of potassium phosphate buffer (pH 7.4), MF1 (200 ng), N A D ÷ (100 nmol) and converting en-
121 zyme (100 ng). The mixture was preincubated for 10 min at 3 7 ° C after which N A D P H (500/~M) and either biliverdin IX a or IX fl (13 #M) were added and the mixture was incubated in a final volume of 200 #1. BvR activity was measured as described above. The increase of the reduction rate of biliverdin IX fl over that of a control (in the absence of either converting enzyme or N A D ÷) measured the presence of MF3. Analytical procedures. Protein concentrations were determined by the method of Bradford[16] using serum albumin as standard. Absorption and fluorescence spectra were recorded at room temperature (18-22°C) on a Hitachi U-2000 double-beam spectrophotometer and on an Aminco Bowman fluorometer, respectively. Polyacrylamide gel electrophoresis. Denaturing SDSpolyacrylamide electrophoresis was essentially performed according to the method of Laemmli [17] using a 3% stacking gel (pH 6.8). The samples were electrophoresed in 11% polyacrylamide resolving gel (pH 8.8) using Bromophenol blue as a tracking dye. The running buffer was Tris-glycine (pH 8.3). The protein samples were routinely prepared by mixing them with the cracking buffer made up of 120 mM Tris-HC1 buffer (pH 6.8), 2% SDS, 20% glycerol and, when indicated, 10% 2-mercaptoethanol. They were heated for 10 min at 100°C. Native gel electrophoresis was performed on 7.5% polyacrylamide gels using the neutral discontinuous buffer system of Williams and Reisfeld [18]. The runs were performed using Tris-barbital buffer (pH 7.5). The electrophoresis was carried out for 30 min at 35 V and then for 4 h at 250 V with Bromophenol blue as a tracking dye. Routinely, 75 ng of pure enzyme was run for silver staining of gels and 185 ng for Western blotting. The gel protein staining was performed either with Coomasie brilliant blue R-250 (0.25% in 50% methanol, 10% acetic acid) or with silver nitrate as described Ref. 10. Chemical modifications of biliverdin reductase. Purified biliverdin reductase (7/~g protein, a single band on native and SDS-PAGE when stained with silver nitrate) was preincubated at 37°C with o-phthalaldehyde in 50 m M Hepes-NaOH (pH 8.0) in the presence of 1% ethanol in a final vol. of 100 #1. Preincubations were performed either at different time periods using 10 mM o-phthalaldehyde, or during a 15 min time period using different o-phthalaldehyde concentrations. At the end of the treatments BvR activity was determined as described above by completing the incubation mixture with biliverdin IX a and N A D P H . When fluorescence was measured, the enzyme preincubated with o-phthalaldehyde was diluted to a final vol. of 0.4 ml and the fluorescence measurements were made with an Aminco Bowman spectrofluorometer. The excitation and emission wavelengths were 337 and 408 nm, respectively.
Succinylation of the enzyme was performed by addition of succinic anhydride (1000 molar excess) to 0.2 nmol (7/~g) of the enzyme in 0.2 ml of phosphate buffer (pH 7.4). The p H of the solution was maintained by addition of M potassium hydroxide. After 30 min the treated enzyme was passed through a Sephadex G-25 column equilibrated with 100 m M phosphate buffer (pH 7.5). When the succinylation was carried out in the presence of either biliverdin or N A D P H , the enzyme was first preincubated with the former for 15 min at 0 ° C and then succinylated as described above. When hydroxylamine was used to probe if succinylation acylated an c-NH 2 of lysine, the former was added to the succinylated enzyme at a 0.1 M final concentration (pH 9.0) and the mixture was incubated at 0 - 4 ° C for 2 h before gel filtration to cleave any O- or S-succinyl derivatives [19]. Modification of the enzyme with 2,3butanedione was performed by preincubating the former (7/~g) in 50 mM borate buffer (pH 8.5) at 37 ° C in the dark, in the presence or absence of the dione. Aliquots of the control and treated enzyme mixtures were then added to the BvR assay mixture at a final 5-fold dilution. When the reaction of butanedione was examined on the enzyme which had been previously preincubated at 0 ° C for 15 rain with either biliverdin, N A D P H or both of them, the reagent was added and then incubated for 10 min at 37°C. Controls were run by using the same procedure but in the absence of butanedione. Determination of the reactive thiol groups. The number of reactive thiol groups of BvR was measured by titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as described by DeLuca and McElroy [20]. The increase in absorbance at 412 nm of a reaction mixture (1.0 ml) containing 7/~g of purified BvR in 0.1 M potassium phosphate buffer (pH 7.4) and 500 # M DTNB, was determined at 25°C against a reference reaction mixture which contained D T N B in buffer. The thiol content was calculated assuming c = 14.1 m M -~- cm -1 at 412 nm for the reaction product. The reaction was complete after 30 min. For determinations performed under denaturing conditions, the enzyme was previously pretreated with 6 M guanidinium hydrochloride. Results
Evidence that different thiol groups are involved in the catalytic activity and in the dimerization of molecular form 1 in liver biliverdin reductase Treatment of MF1 of rat liver biliverdin reductase with p-chloromercurisulphonate, iodoacetamide, N E M and N P h M at various concentrations showed that the inhibition of the conversion of MF1 into MF3 was achieved at lower concentrations and shorter incubation times than the inhibition of the enzymatic activity (Fig. 1). p-Chloromercuribenzoate inhibited 90% of the conversion at concentrations where only 15% of the en-
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were performed for 15 rain at 37°C. (B) Time-course of the loss of the reductase activity (o), and increase in fluorescence intensity (e) of biliverdin reductase, MFI. The enzyme was treated with 10 mM o-phthalaidehyde for the indicated times. Parallel experiments for measuring activity and fluorescence were run. The data were presented as Ft - Fo vs. time, where Ft represents the fluorescence at the time t and F0 at time to. The excitation and emission wavelengthswere 337 nm and 408 nm, respectively.
126 cence (48%). It is obvious that at a given concentration of o-phthalaldehyde the loss of enzymatic activity parallels a corresponding increase in the relative fluorescence.
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Inactivation of biliverdin reductase by butanedione and protection by substrates It is known that guanidinium groups of arginine can function as contact sites for the anionic phosphate groups of the pyridine nucleotides [22]. The same basic group could also stabili7e, by electrostatic binding, the carboxylate anions of the propionate side chains of biliverdin. It followed that the reaction of the enzyme with butanedione could prove the possible involvement of arginine residues in its activity. As can be seen in Fig. 5, butanedione inactivated the enzyme both in a concentration- and in a time-dependent manner. The inhibition by butanedione was completely countered by preincubation of the reductase with N A D P H , while biliverdin did not affect the inactivation of the enzyme by 10 or 15 m M butanedione (data not shown). Therefore, at least one arginine residue is in the close proximity of the N A D P H binding site, but removed enough from the biliverdin binding site. This arginine residue could therefore be the stabilizing cation of the phosphate residues which are nearer to the adenine moiety of the nucleotide.
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Spectroscopic evidence for the presence of a positively charged ion at the binding site of NADPH It is known that the UVm~ of N A D P H at 340 nm shifts to 325 nm when the latter is complexed with horse liver alcohol dehydrogenase, bovine heart lactate dehydrogenase or rat liver lactate dehydrogenase [23]. A similar spectroscopic shift was observed when 3-acetyl
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Fig. 6. Spectral shifts of NADPH and 3-acetyl NADPH on bindin8 to biliverdin reductase. (A) Spectra of ( - - - - - ) NADPH when added t o bovine serum albumin (1 m s / m l ) in 20 mM phmphate buffer (pH 7.4) and ( ) NADPH added to bih'vexdin redu~tase (160 ttg/ml) in the same buffer. (B) Spectra of ( - - - - - - ) 3-acetyl-NADPH and ( ) 3-acetyi-NADPH plus b i l i v e r d i n - ~ . The 3-acetyl-NADPH w a s obtained from 3-acetyl-NADP+ by chemical reduction with dithionite. Inset of (B) shows the difference spectrum between ( ) and ( - - - - - - ) of the last spectra.
127 about 3/~ from the dihydropyridine ring would increase the observed transition energy for light absorption by the ring and account for the aforementioned shifts. The spectra of the NADPH complex showed a UVm~ at 327 nm, while NADPH itself as well as NADPH in the presence of BSA showed the expected maximum at 340 nm (Fig. 6A). The 3-acetyl NADPH-MF1 complex showed a UVm=, at 348 pm while 3-acetyl NADPH itself or in the presence of BSA gave the expected UVmx at 360 nm (Fig. 6B). The differential spectra at two different nucleotide concentrations can be seen in the inset to Fig. 6B. We have already shown that 3-acetyl NADPH is a good co-substrate for biliverdin reductase [8]. These spectral data provided evidence for the presence of a positive charge in the vicinity of the binding site of NADPH in the reductase similar to those present in the dehydrogenases. Crystallographic data have shown the presence of arginine guanidinium cations in the active sites of lactate dehydrogenase as well as of glyceraldehyde-3-phosphate dehydrogenase [25], while in horse liver alcohol dehydrogenase the positive charge is a.pparently related to the Zn2÷ atom located about 3-4 A from the nicotinamide ring [26]. In the case of biliverdin reductase, the positive charge very
likely resides on the nitrogen of the alkylammonium ion formed by the E-amino group of the lysine residue which was shown to be closed to the substrate binding thiol residue (see above). In NADH-cytochrome b5 reductase, a cysteine [27] and a lysine residue [19] were shown to be involved in NADH binding.
Inactivation of bilioerdin reductase by trypsin and protection by substrates The presence of lysine and arginine residues at or near the active center of the reductase called for an examination of the effect of trypsin on the activity of molecular form 1 of the enzyme. A 2 mM trypsin concentration inactivated 70% of the enzyme at 5 min and 90% of the enzyme after 11 rain (Fig. 7). Both the substrate and the co-substrate strongly protected the enzymatic activity and in the presence of each of them 80% of the activity was preserved after a 10 rain preincubation with trypsin (Fig. 7). These results could be expected from an enzyme with a lysine residue at the active site and with an arginine residue needed for the stabilization of the co-substrate. The protection by either substrate or co-substrate suggest that the fast attack by trypsin takes place at the lysine (or arginine) residues at the active center and this is supported by the fact that the degraded reductase did not shift the pyridine nucleotide spectra any further. Discussion
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TIME, rain Fig. 7. Time dependent inactivation of biliverdin reductase by trypsin. MF1 (2/~M) was treated with trypsin (2 mM) in the absence (O) and presence of either (,,) biliverdin (11 btM) or (zx) N A D P H (500/*M). The enzyme was preincubated 10 rain at 370C with each of the substrates before trypsin was added. After the preincubation with trypsin during the indicated times, soybean trypsin inhbitor (4 raM) was added and the activity was assayed as described in Experimental procedures.
An analysis of the structural features of molecular form 1 of biliverdin reductase will be helpful to understand its dynamics when the primary sequence and crystallographic data will outline its architecture. The reduction of biliverdin to bilirubin by a hydride transfer from a reduced nicotinamide ring bears similarities to the reduction of carbonyl groups by the dehydrogenases. It has been repeatedly suggested that in the latter a sp2-sp 3 transition takes place during catalysis [221. The reversible addition of thioles to the C-10 methine of biliverdin and to carbonyl groups have similar thermodynamic and kinetic parameters for the equilibrium reaction [14]. It is therefore conceivable that there is a convergence of the active center geometries of some dehydrogenases and of biliverdin reductase. It is known that in glyceraldehyde-3-phosphate dehydrogenase a thiol residue is involved in the hydride transfer to the carbonyl group, very likely through a prior addition of cysteine to form a tetrahedral intermediate [22]. We have shown that a thiol residue present at the active center binds to biliverdin (Table I). Hydride transfer to the C-S bond will result in the fast cleavage of the adduct with formation of bilirubin. The finding that a lysine residue is located at about 3 ,~ from the thiol group at the active center (Fig. 4 and Table II) and the
128 Pyridoxal phosphate and biliverdin also conferred full protection to the enzyme from alkylating agents, very likely due to the presence of a lysine residue at the active center. This is supported by the formation of an isoindol derivative in the presence of o-phthalaldehyde which could only result from the reaction of neighboring thiol and c-NH 2 residues with the reagent. The cationic ion formed from the c-NH 2 of the lysine residue could be responsible for the hypsochromic shift observed in the UVm~, of the reduced nucleotide (NADPH and 3-acetyl-NADPH) when they bind to the enzyme. The protection of the enzymatic activity in the presence of NADPH from chemical modification by butanedione suggest that arginine residues are involved in salt-like linkages with phosphate residues of the co-substrate.
/ Fig. 8. A model of the ternary transition-state complex of biliverdin reductase MF1.
possibility that its c-NH 2 is present in the form of an ammonium ion at a 3-4 A distance from the nicotinamide ring allows us to outline a picture for the transition state of the enzyme-co-enzyme substrate ternary complex (Fig. 8). The evidence that an arginine residue is also essential for enzymatic activity and is placed at the binding site of NADPH could also be the origin of the UV~, shift observed for the pyridine nucleotides after binding to the enzyme (Fig. 6). In the absence of crystallographic data it is difficult to decide on this issue, since either the lysine or arginine residues could be attached to flexible loops which undergo conformational changes during catalysis. These residues could move into the active center during the formation of the ternary complex (see Ref. 25). We favor the location of the lysine residue near the binding site of biliverdin and NADPH rather than the arginine residue since biliverdin does not prevent the inactivation of the enzyme by butanedione.
Conclusion Of the three thiol residues present in MF1 of biliverdin reductase only two could be modified with alkylating agents under nondenaturing conditions. The more exposed thiol group is that involved in the conversion of MF1 into MF3 of the liver reductase. The thiol group involved at the active site in the binding of biliverdin is less reactive. This binding was not affected by guanidinium chloride and results very likely from the formation of a C-S bond. Full protection of the enzyme from alkylating agents could only be achieved by the simultaneous presence of both biliverdin and NADPH.
Acknowledgements This work was made possible by grants (GM-11973) from the National Institutes of Health and from CONICET (Argentina).
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129 18 Williams, P.E. and Reisfeld, R.A. (1964) Ann. N.Y. Acad. Sci. 121, 373-381. 19 Loverde, A. and Strittmatter, P. (1968) J. Biol. Chem. 243, 57795787. 20 De Luca, M. and McElroy, W.D. (1966) Arch. Biochem. Biophys. 116, 103-107. 21 Purl, R.N., Bhatnagar, D. and Roskoski, Jr. R. (1985) Biochemistry 24, 6499-6508. 22 Jeffrey, J. (1982) in Stereochemistry (Tamm, Ch., ed.), pp. 113-160 (and references therein), Elsevier, Amsterdam.
23 Van Eys, J., Stolzenbach, F.E., Sherwood, L. and Kaplan, N.O. (1958) Biochirn. Biophys. Acta, 27, 63-83. 24 Kosower, E. (1962) Molecular Biochemistry, pp. 213-219, McGraw Hill Series in Advanced Chemistry. 25 Garavito, R.M., Rossmann, M.G., Argos, P. and Eventoff, W. (1977) Biochemistry 16, 5065-5071. 26 Eklund, H., Samarma, J.P. and Jones, T.A. (1984) Biochemistry 23, 5982-5996. 27 Hackett, C.S., Novoa, W.B., Ozols, J. and Strittmatter, Ph. (1986) J. Biol. Chem. 261, 9854-9857.
