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Further studies on the electron transport proteins involved in the oxidative decarboxylation of glycine
ARCHIVES
OF
Further
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
on the Electron Oxidative
1”ARIETTA Department
of Biochemistry,
703-711 (1967)
ii?&
Transport
Decarboxylation L. BAGINSKY
Scripps
Clinic
Received
AND
Proteins
in the
of Glycine’ F. R/I. HUENNEKENS
and Research Foundation, December
Involved
La Jolla,
Cali)ornia
92037
20, 1966
The oxidative decarboxylation of glycine is catalyzed by a system consisting of 4 proteins isolated from Peptococcus glycinophilus. Ps, a small molecular weight, dithiolcontaining protein, and Ps, a flavoprot’ein, are concerned with electron transfer from glycine to DPN (i.e., glycine -+ Pz + P1 + DPN). Reversed electron flow through this sequence is also demonstrable (DPNH --) PI + Pp -+ DTNB). From its ability to be reduced by either DPNH or dihydrolipoic acid, and changes in absorption spectrum when the reduced enzyme is treated with arsenite or mercurials, PI appears to be a lipoyl dehydrogenase. Consistent with this hypothesis, lipoyl dehydrogenases from pig heart and Escharichia coli can replace Pa in each of the above transfer sequences.
Reversibility of this electron transport sequence was demonstrated by coupling the oxidation of DPNH to the chromogenic acceptor, 5,5’-dithiobis(2-nitrobenzoic acid), according to Eq. (3):
Sagers and Gunsalus (1) first demonstrated that, cell-free extracts of Peptococcus glllcinophilus were able to catalyze the oxidative decarboxylation of glycine according to the interesting stoichiometry of Eq. (1) : Glycine + tetrahydrofolate + DPN+ 4 5, lo-methylene tetrahydrofolate + CO, + NHd+ + DPNH.
DPNH + P3 ---f PZ + DTNB.2 (1)
Studies by Klein and Sagers (2) and by the present authors (3) have demonstrated that the enzyme system responsible for the above reaction can be resolved into four components: PI, a pyridoxal phosphate-containing protein; Pe, a heat-stable protein; PD, a flavoprotein; and P4, an uncharacterized protein. In a recent communication (3) we have proposed a mechanism for reaction (1) in which oxidation at the a-carbon of glycine is linked to DPN via fractions P2 and Ps, as shown schematically in Eq. (2) : Glycine + PZ -+ P3 + DPN.
(2)
1 Paper IX in the series “Flavin Nucleotides and Flavoproteins.” This work was supported by grants from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health (AM 08398), and the American Heart Association (63-G-146 and 66-796).
(3)
PZ is a low molecular weight, heat-stable protein whose properties and function closely resemble those of thioredoxin, an oxido-reduction component of the ribonucleotide reductase system in Escherichia coli (4). Thioredoxin contains a disulfide group that is reduced to a vicinal dithiol by a TPNHdependent flavoprotein (5). Since P, likewise undergoes reversible oxidation-reduction in the systems represented by Eq. (2) and (3), we have previously proposed that P, also has a functional disulfide group that is reduced by Pa, a DPNH-dependent flavoprotein (3). The present communication provides further evidence in support of the roles of P? and P3 as oxido-reduction carriers intervening between glycine and DPN (reaction 1). Inhibitor studies are consistent with the presence of a functional disulfide bridge in Pt. P3 has been shown to possess many of the characteristics of a lipoyl dehydrogenase; conversely, lipoyl dehydrogenases from other
703
704 sources can replace overall reaction.
BAGINSKY
P, as a component
AND
in
the
IIUE?U’NEKENS
amide, or by redllction of AP-DPN (increase in absorbancy at 363 mp) with dihydrolipoic acid. In addition to t.he substrate and ppridine nucleoEXPERIMENTAL PROCEDURE tide, incubat,ion mist.ures co11taiued ph0sphat.e buffer, EI)TA, bovine serum albllmill, and cataMale&&. The following chemicals were oblytic amounts of the flavoprotein. tained from commercial sources: DPN, DPNH, In all of the above assays, substrates and et>pyridoxal phosphate, DTNB, dihydrolipoic acid, zymes were incubated for 2 minutes at 38” before lipaamide, and pig heart lipoyl dehydrogenase starting the reaction. When reversed electron flow (Sigma Chemical Co.) ; 3-acetylpyridine-DPN was studied, DTNB was added after the preincu(P. L. Biochemicals) ; N-ethylmaleimide (Sehwarz bation. Reactions were started bv adding the Bio-Research, Inc.) ; iodoacetamide (California pyridine nucleotide to t,he experimental cuvette. Corporation for Biomedical Research); and a The blank cuvette contained the same components standard solution of bovine serum albumin (Aras the experimental, except for the nucleotide. In mour Pharmaceutical Co.). Iodoacetamide was all cases, blanks without substrate were also run. recrystallized from methanol-water before use. Absorbancy changes were followed at 15-second A highly purified sample of lipoyl dehydrogenintervals for periods up to 4 minutes in a Beckase from E. coli was generously provided by Dr. man Spectrophotometer, model DU, at 38”. The C. H. Williams, University of Michigan. Tetrahyexact composition of the assay mixtures is given drofolate was a gift from Dr. V. S. Gupta of this in the legends of Tables and Figures. department. Hydroxylapatite gel was prepared Preparation of enzymes. Peptococcus glycinaccording to the procedure of Levin (6). ophilus was grown anaerobically in the medium Methods. Absorption spectra were determined of Sagers and Gunsalus (I), modified as described at room temperature (22-25”) with a Beckman previously (3). Protein fractions (PI, P,, Ps, and DK-2 recording spectrophotometer. Thunberg Pa) were separated from each other and purified cuvettes with the side arm replaced by a rubber by the procedure summarized in a previous comcap were used in the anaerobic experiments. The munication (3). The following is a typical prepacuvettes were evacuated and flushed with preration: Fifty-five gm of frozen cell-paste was purified Nz six to ten times; additions were made thawed and suspended in 165 ml of 0.02 M potasby injecting solutions through the rubber cap. sium phosphate buffer, pH 7.0, containing 0.01% Protein was determined by the biuret method of Na2S. Fift.y-ml aliqnots of this cell suspension Gornall et al. (7) with bovine serum albumin as were sonicated for 20 minutes in a Raytheon 10 standard. KC sonic oscillator cooled with circulat,ing wat,er Assay systems. For the determination of reacat -5”. Intact cells and debris were removed by tion (I) reduction of DPN (or AP-DPN)2 was folcentrifugation for 30 minutes at 17,000 rpm in the lowed spectrophotometrically at 340 rnp (or 363 SS-34 rotor of a Servdl RC-2 centrifuge. (Unless mF) in a system containing glycine, phosphate otherwise stated, all subsequent centrifugations buffer, pyridoxal phosphate, tetrahydrofolate were performed in the same manner.) The superplus mercaptoethanol, catalytic amounts of all natant fluid (182 ml, 24.8 mg protein/ml) was four enzymes, and DPN (or AP-DPN). Reversed kept frozen overnight. Protamine sulfate (22.6 electron flow (reaction 3) was studied by following the reduction of DTNB at 412 w in a system ml of a 2% solution) was added dropwise and the containing phosphate buffer, EDTA, catalytic pH of the mixture was adjusted to pH 6.7 by t)he addition of 1 N KOH. After the preparation had amounts of PZ and PI, and DPNH. DTNB solubeen immersed in an ice-bath for 20 minutes, t,he tions were prepared just prior to use by dissolving 10 #moles of DTNB in 1 ml of ethanol and dilut,ing precipit,ated nucleic acids were removed by cento a volume of 10 ml with 0.5 M Tris-chloride trifugation for 30 minutes. To the supernatant, (200 ml) 467 ml of ammonium sulfate buffer, pH 8.0. Two-tenths ml of this solution was solution used for assay systems in which the total volume solution saturated at 5” (pH 7) was slowly added. was 1.0 ml. After standing at 2-5” for 30 minutes, the precipiLipoyl dehydrogenase activity was determined tate (@70y0 ammonium sulfate fraction) was disby measuring either DPNH oxidation (decrease carded. Solid ammonium sll1fat.e (120 gm) was in absorbancy at 310 rnp) in the presence of liposlowly added to the supernatant fluid (467 ml) to obtain a satllrated solution. The solution was z Abbreviations used: DTNB, 5,5’-dithiobisstirred for one hour at 2-5’ and then centrifuged (2-nitrobenzoic acid) ; AP-DPN, 3.acetyl-pyridine for 45 minutes. The precipitat,e was dissolved in analogue of DPN; NEM, Nethylmaleimide; IAA, 0.02 M potassium phosphate buffer, pH 7.0 (7(r iodoacetamide; PCMS, p-chloromercuriphenyl 100% ammonium sulfate fraction) sulfonate. The above solution (20 ml) was chromato-
OXIDATIVE
DECAR~OXYLATION
graphed on a 4.5 X 42-cm column of Sephadex G-50 (fine) equilibrated with 0.02 M potassium phosphate buffer, pH 7.0. Elution was carried out with the same buffer at a rate of 12 drops per minute; 5-ml fractions were collected. The first protein fraction eluted was yellow-green, and this was followed by a yellow-brown fraction. Rubredoxin, ferredoxin, and free flavin remained on the column. Those tubes containing the yellow-brown fraction plus the last tube of the yellow-green fraction were pooled and lyophilized. This sample contained the bulk of the Pp activity. Those tubes containing the yellow-green fraction were pooled and concer,trated by ultrafiltration. The latter fraction, which contained P,, Pa, and P*, along with some Ps, could be stored frozen for 2 weeks without apparent loss in activity. For further purification of PZ, the lyophilized fraction was dissolved in 20 nil of water and heated for 3 -minutes in a boiling water bath. The sample was immediately cooled in an ice-bath and the precipitate was removed by centrifugation for 20 minutes at 40,000 rpm (No. 40 rotor) in a Spinco ultracentrifuge. The supernatant solution was dialyzed for 6 hours against 4 liters of 0.02 M potassium phosphate buffer, pH 6.8 (one change of buffer), and concentrated by the use of dry Sephadex G-25. Two such fractions were pooled (32 ml, 5 mg protein/ml) and chromatographed on a 2.8 X 4:1-cm column of hydroxylapatite that had been equilibrated with 0.02 M potassium phosphate buffer: pH 6.8. Gradient elution was performed with potassium phosphate buffer, pH 6.8 10.05 -+ 0.2 M, 800 ml each). The flow rate was 11 drops per minute and fractions of 80 drops were collected. Two principal protein peaks3 were obtained: a salmon-colored fraction in tubes 68-85 and a colorless fraction (slightly yellow in very concentrated solutions) in tubes 90-120. PZ activity was found in both peaks, but the specific activity of the colorIess fraction was four times that of the other. Tubes 90-120 were pooled, concentrated with dry Sephadex G-25, and kept frozen until use. For the separation of P1, I’s, and P4, the concent.rated yellow-green fraction described above (50 ml, 25 mg prot.ein/ml), was chromatographed on a 3.5 X 4O-cm column of D~AE-cellulose that had been equilibrated with 0.02 M potassium phosphate buffer, pH 7.0. Gradient elution was performed with potassium phosphate buffer, pH 7.0 (0.05 --t 0.4 M, 800 ml each). Five-ml fractions were collected at a rate of 15 drops per minut,e. The first peak contained the Pt activity, t,he see3 Klein and Sagers (8) have previously reported the separation of Pz into two peaks of activity by chromatography on the same adsorbant.
706
OF GLYCINE
ond peak was discarded, and P1 and P) were found in the third and fourth pea.ks, respectively. Solutions containing each of these activities were pooled, concentrated by ultrafiltration, and kept frozen. Each of the above three fractions was purified further by chromatography on hydroxylapatite. A 3.5 X 20-cm column, equilibrated with 0.02 M potassium phosphate buffer, pH 6.8, was used for 150 mg of each fraction. Elution was performed with a linear gradient of potassium phosphate buffer, pH 6.8 (0.05 + 0.3 M, 800 ml each). The flow rate was 12 drops per minute. Solutions containing the desired activities were pooled, concentrated by ultrafiltration, and stored frozen. Pt was further purified by ammonium sulfate precipitation (70-77% fraction). This fract,ion was dissolved in 0.05 M potassium phosphate buffer, pH 7.2, and used directly, or desalted by passage through Sephadex G-25. At this stage of purification each of the four proteins was not contaminated with any of the other three. Pq gave a single band when examined by electrophoresis on acrylamide gel, but each of t’he other fractions displayed, in addition to the principal band, several other weak contaminating bands. A somewhat different procedure for the purification of Pt and Pz has been used by Klein and Sagers (S), and recently, the same authors have described an excellent new procedure for the separation of the four proteins (9). RESULTS
AND
~eq~~~~~~.~n~ for
DISCUSSION
the olierall
r~~et~~.
As
shown by Klein and Sagers (9) and also by TABLE REDUCTION
OF DTNB
I BY GLYCINE
Componenl.omitted None” Glycine Tetrahydrofolate Pt P*
21.2 0.3 0.3 0.3 3.8
aThe complete system contained in 1.0 ml: lOOpmoles of phosphate buffer, pH 7.0; 75 mpmoles of pyridoxal phosphate; 2 pmoles of tetrahydrofolate; 4 rmoles of glycine; 0.2 #mole of DTNB (in ethanol-Tris buffer as described in the Experimental Section); 0.8 mg of PI; 0.25 mg of Pp; and 0.4 mg of Pg. Blank cuvettes contained all components except Pz. b Based upon E = 13.6 X lo3 M-’ cm-’ at 412 ml* (lo).
BAGINSKY
706 TABLE DPNH-DEPENDENT Component omitted
None0 PZ PP DPNH
AND
II
REDUCTION
OF DTNB DTNB reduced (mpmoles/min)
10.6 0.01 0.03 0.00
a The complete system contained in 1.0 ml: 50 pmoles phosphate buffer, pH 7.2; 10 pmoles of EDTA; 0.2 pmole of DTNB (in ethanol-Tris buffer); 0.3 rmole of DPNH; 0.03 mg of P,, and 0.01 mg of Pa. For the first three experiments, the blank cuvettes contained all components except DPNH. When DPNH was the component omitted, P2 was deleted from the blank.
our studies (3), the oxidative decarboxylation of glycine linked to DPN (Eq. I), requires four protein fractions (PI, Pg, Pa, and P4) in addition to tetrahydrofolate and pyridoxal phosphate as cofactors. The requirement for tetrahydrofolate is absolute, while that for pyridoxal phosphate is partial and variable depending upon the extent to which PI is present as the apoenzyme. E~ta~lishe~nt of Pz and P3 as orido-yeduetion carriers between ylycine and DPN. In the mechanism that we have presented previously (3), it was postulated that oxidation and decarboxylation of glycine occur as a concerted reaction, and that the reducing power generated in this step is carried to DPN via Pt and Pt (cf. Eq. 2). Support for the placement of P, prior to P3 in the electron flow from glycine to DPN was provided by the observation, illustrated as Fig. 1 in our previous paper (3), that dithionite-reduced P8 is rapidly oxidized by DPK, but not by TPN; this specificity for pyridine nucleotide is in accordance with that noted for the overall reaction. In addition, oxidation of glycine can occur in the absence of P3 and DPN provided that an external electron acceptor, such as DTNB, is present (Table I). All of the other components of the complete system (Eq. 1) are required for the DTNB-linked oxidation of glycine. Although not indicated in Table I, omission of PZ completely abolished the reduction of DTNB; mixtures without Ps could be used, therefore, as blanks.
HUENNEKENS
Reversed electron $ow. It was also convenient to study the function and relation ship of P, and P, in the reverse sequence, i.e., by generating the reducing power from DPNH, rather than glycine. As described by Eq. (3), oxidation of DPNH can be coupled to the reduction of DTNB in the presence of catalytic amounts of Pz and Ps (Table II). This experiment also provided evidence that each of these proteins was free from contamination by the other. As predicted by the sequence of Eq. (3), DPNH can be used to reduce substrate amounts of PB (Fig. 1). The absorption band of P, in the 400-500 rng region, characteristic of oxidized flavoproteins, was replaced by a lower and more broad band at a somewhat lower wavelength when progressively larger amounts of DPNH were added. At the same time the absorbance between ,500 and 700 mp increased, probably due to formation of a charge-transfer complex between the flavin semiquinone and DPN. This point will be discussed further in a later section of the paper. Under these conditions, the solution in the cuvette changed from yellow to red. Similar results have been reported for other flavoproteins reduced by their substrates (11, 12). Complete reduction of Pa, as indi-
300
400 500 600 Wavelength,mp
700
FIG. 1. Reduction of Pa by DPNH. The experimental cuvette contained in a final volume of 1.0 ml: 5 mg of P1; 70 pmoles of phosphate buffer, pH 7.3; 2 rmoles of EDTA; and 40 pmoles of ammonium sulfate. Oxidized Pa (p); plus 0.01 ml of 10 mu DPNH (- - -); plus an additional 0.03 ml of DPNH (. . . . ); opened to air plus addition of dithionite (-.-.). For the first two spectra the blank cuvette contained water; 1 ml water plus 0.02 ml of 10 mM DPNH was used for the blank in the latter two spectra. Additions of DPNH were made under anaerobic conditions.
OXIDATIVE
DECARBOXYLATION
I
300 400 500 600 70~? Wavelength,mp FIG. 2. Oxidation of reduced Pa by Pt. The experimental cuvette contained in a final volume of 1.0 ml: 5 mg of P3; 70 pmoles of phosphate buffer, pH 7.3; and 2 Mmoles of EDTA. Oxidized Pa (+-) ; reduced with dithionite (under N2) (-.-. ) ; plus 0.02 ml of PZ (17.7 mg protein/ml) (. . . .); plus an additional 0.02 ml of P, (- - -). Water was used as the blank for the first three spectra while 1.0 ml of water plus 0.04 ml of PS was used for the final spectrum. Additions of P2 were made under anaerobic conditions.
cated by abolition of the 450 and 550 rnp bands, was obtained by the addition of dithionite. Again, from Eq. (3), it should be possible to oxidize reduced P3 with substrate amounts of P,. This is demonstrated in Fig. 2. When P, was fully reduced with dithionite and then treated with successively larger amounts of oxidized Pz, the absorbancy at 455 rnp returned to a value close to that of the fully oxidized enzyme, while that in the 500-600 rnp region was higher than the original absorbancy. In this experiment the P2 used was assumed t’o be in the oxidized for+ as isolated; this assumption is supported by observations described below. Finally, oxidation of DPNH can be linked to PZ, provided that PB is present in catalytic amounts. The experiments described in the next section make use of this reaction to prepare substrate amounts of reduced Pz. Evidence that Pp contains a functional dithiol group. The possibility that the oxidoreduction component of PZ is a vicinal dithiol group, sitiilar to that occurring in thioredoxin (4), was investigated with the use of sulfhydryl inhibitors. As shown in Table III, iodoacetamide and NEM inhibited the reduced form. of Pz but not the oxidized form.
707
OF GLYCINE
In these experiments P, was reduced enzymically (by incubation under anaerobic conditions with DPNH and P3) and then incubated with iodoacetamide or NEM under anaerobic conditions. Exce& inhibitor was removed4 by passage of the mixture through Sephadex G-25. A control sample was treated in the same manner except that water was added instead of the inhibitor. Activity of P, was then determined by measuring DTNB reduction in the presence of additional PB and DPNH. Experiment 1 in Table III shows that samples of P, treated with either iodoacetamide or NEM were almost inactive when compared with the untreated control. That the observed effect was not caused by inhibition of P, rather than PZ, due to inTABLE INHIBITION
OF AND
Experimalt NO.
REDUCED
III Pz
BY
IODOACETAMIDE
N-ETHYLMALEIMIDE~
Sample
DTNB reduced (U moles/ min)
Ps used Specific in assay activity* bw)
1
Reduced Reduced Reduced
PZ Pp + IAA Pz + NEM
28.1 0.66 0.66
0.082 0.078 0.079
329 8 8
2
Reduced Oxidized Reduced
Pz Pz + NEM P, + NEM
31.7 40.6 0.96
0.090 0.089 0.090
338 436 1
u Under anaerobic conditions Pt (3.54 mg) was reduced by treatment with 2.5 pmoles of DPNH, 0.15 mg of Pz, and 50 Mmoles of potassium phosphate, pH 7.2 (total volume, 0.65 ml) for 15 minutes at 38”, and then incubated for 60 minutes at 38” wit,h either 10 mM iodoacetamide or 4 mM NEM. The preparation was then passed through a 1 X 20.cm column of Sephadex G-25 that had been equilibrated with 0.05 M phosphate buffer, pH 7.0; elution was accomplished with the same buffer. An aliquot of P, was assayed for activity in a system that contained in 1.0 ml: 50 pmoles of phosphate buffer, pH 7.2; 10 rmoles of EDTA; 0.2 rmole of DTNB; 0.3 pmole of DPNH; 0.044 mg of PI; and amounts of treated P? as indicated. * mpmoles DTNB reduced/min/mg protein. 4 Addition of the inhibitors directly to the assay system (DPNH + Pt --t Pp + DTNB) would have not revealed which of the components had been inactivated.
705
BAGlNSKY
0.01
0.02
0.03
0.04
AND
0.05
Flaw, mpmoles FIG. 3. Lipoyl dehydrogenase activity of Ps. Assay mixtures contained in 1.0 ml: 60 pmoles of phosphate buffer, pH 7.3; 2 pmoles of EDTA; 2.4 pmoles of dihydrolipoic acid; 0.6 mg of bovine serum albumin; 0.8 /*mole of AP-DPN; and varying amounts of the enzymes as indicated. Concentration of each enzyme is expressed in terms of flavin content. Flavin was determined by measuring the decrease in absorbancy at 455 rnp following addition of dithionite and using a differential extinction coefficient of 11.3 X lo3 MM' cm-l.
complete removal of the inhibitor, was borne out by Experiment 2. When both t,he oxidized and reduced forms of Pz were incubated with NEM, only the latter was inhibited. Repetition of this experiment several times showed that treatment of the oxidized form with NEM occasionally produced a stimulation of the activit,y. When the above experiment was repeated with 10 mM Cd++ or arsenite as inhibitors (at pH 5.0 to prevent precipitation of Cd++), the results were less conclusive. In both cases the observed inhibition was only 3040 %. The failure of these reagents to completely inhibit Pz, as would be expected for a vicinal dithiol, may be due to dissociation of the enzyme-inhibitor complexes upon passage through Sephadex.5 Relationship of Pa to lipoyl clehydrogenases. The absorption spectrum of P, in the visible region is very similar to t,hat of a lipoyl dehydrogenase (cf. Fig. 2 of Ref. 13). It was not surprising, therefore, to find that P3 5 Arsenite (1W M) and CdCl, (lo* M), when added directly to the assay system, inhibited the overall reaction (Eq. 1) by 72 and 739&, respectively.
IIUENNEKENS
exhibited good lipoyl dehydrogenase activity lvhen tested eit,her by reduction of Al’-DPN with dihydrolipoic acid or by reduct.ion of lipoamide with DPJSH. The data of Fig. 3 compare the ability of Pa, pig heart lipoyl dehydrogenase, and E’. coli lipoyl dehydrogenase to catalyze the reduction of AP-DPN by dihydrolipoic acid; each enzyme was tested at several levels based on flavin eoncentration. Although the pig heart’ enzyme exhibited the greatest activity, Pa was also an effective lipoyl dehydrogenase. Similarly, the pig heart enzyme and P, showed the same activity in catalyzing the DPNHdependent reduction of lipoamide. Lipoyl dehydrogenases are known to be inhibited by DPNH but not by AP-DPNH. Since Ps, when tested as a lipoyl dehydrogenase, n-as also sensitive to DPNH, it seemed likely that our earlier data, in which the overall reaction (Eq. 1) was characterized by a decline in rate with t,ime, would have been consistent with inhibition by one of the products, DPNH. As shown in Fig. 4, AP-DPN not only replaces DPN in the
Time, min. FIG. 4. Comparison of DPN and AP-DPN in the glycine oxidation system. The assay mixtures contained in a volume of 1.0 ml: 75 pmoles of phosphate buffer, pH 7.0; 1 pmole of tetrahydrofolate; 25 pmoles of mercaptoethanol; 75 mpmoles of pyridoxal phosphate; 4 pmoles of glycine; 0.35 mg of protein (PI + Pz + Pa + P4) and 0.8 pmole of DPN or AP-DPN.
OXIDATIVE
DECARBOXYLATION
Wavelength,mp FIG. 5. Reduction of PB by dihydrolipoic acid. The experimental cuvette contained initially the same components as described in the legend for Fig. 1. Oxidized Pa (---); plus 0.01 ml of 12 mM dihydrolipoic acid (- - -) ; plus an additional 0.93 ml of dihydrolipoic acid (. .); opened to air, plus dithieonite (-.-.). Additions of dihydrolipoic acid were made under anaerobic condit.ions.
overall assay system but also abolishes the decline in activity seen with the former. It was shown previously (l;ig. 1) that DPNH can reduce I’$ to a red, presumably scmiquinone, stage. The same result was obtained when P)3was reduced anaerobically Lvith dih,ydrolipoic acid (Fig. 5). Even upon addition of a large excess of dihydrolipoic acid (0.96 pmole per 0.0‘25 pmole of flavin) the enzyme \vas not reduced beyond the scmiquinone form; complete redu&ion was obtained, however, with dithionite. In agreement with the findings of Massey and Veeger (14)) almost identical results were obtained with the pig heart enzyme. In contrast, the E. co&i enzyme gradually assumes the fully reduced form as the concentratjion of substrate is increased (13). The above findings prompted us to test P, for other characteristics that have been reported for pig heart and E. coli lipoyl dehydrogenases and yeast glut#athione reductase. Massey and his co-workers (11-14) have proposed that the above enzymes contain a disulfide bridge which, upon reduction, permit
the
transfer
of one electron
from
one of
the sulfhydryl groups to bhe flavin; interaction of the resultant sulfur radical with the flavin semiquinone would stabilize both groups. This proposed complex is characterized by an absorption maximum at about 530 rnp, and its interaction with DPNH is
709
OF GLYCINE
believed to form a charge-transfer complex with absorption maximum at about 570 rnp. Formation of the latter explains the broader absorption band above 500 rnp observed when the above enzymes are reduced with DPNH. The same results have also been obtained with P3 (cf. Figs. 1 and 2). Also, as found by the above authors, addition of PCMS to the DPNH-reduced enzymes resulted in complete reduction of the flavin; the same result is observed with DPNHreduced P3 (Fig. 6). Finally, as reported for the lipoyl dehydrogenases and the yeast glutathione reductase, addition of increasing amounts of DPNH to the arsenite-treated enzyme resulted in progressive bleaching of the 455 rnp band accompanied by formation of a green band with absorption maximum at 720 rn#. The latter has been attributed to the formation of a charge-transfer complex between the fully reduced enzyme and DPlS. A
400
500
600
700
800
Wavelength, mp
FIG. 6. Effect of PCMS on DPNH-reduced Ps. The experimental cuvette contained in a total volume of 1.0 ml: 80 pmoles of phosphate buffer, pH 7.3; 2 rmoles of EDTA; and 11.9 mg of Pa. Oxidized PB (blank: water) (--) ; plus 0.02 ml of 20 rnM DPNH (blank: 1 ml water plus 0.01 ml of DPNH) (- - -); plus an additional 0.02 ml of DPNH 1 hour later (blank: plus an additional 0.01 ml of DPNH) (. .); plus 0.02 ml of 50 mM PCMS added to both experimental and blank cuvettes (-.-.). Additions of DPNH and PCMS were made under anaerobic conditions.
710
BAGINSKY
AND
Wavelength,q FIG. 7. Reduction of arsenite-treated Pa by DPNH. The experimental cuvette contained in a total volume of 1.0 ml: 80 pmoles of phosphate buffer, pH 7.3; 2 Gmoles of EDTA; 1 pmole of sodium arsenite and 9.85 mg of PI. The blank cuvette contained the same components with the exception of the enzyme. Oxidized Ps (--); plus 0.01 ml of ‘20 mM DPNH (blank: plus 0.004 ml of DPNH) (- - -); plus an additional 0.01 ml of DPNH (blank: plus an additional 0.004 ml of DPNH) (-.-.); plus an additional 0.02 ml of DPNH (blank: plus an additional 0.01 ml of DPNH) (...-). Additions of DPNH were made under anaerobic conditions.
comparable experiment, involving Pa, is shown in Fig. 7. Possible replacement of P3 by lipoyl dehydrogenase and of P9 by dihydrolipoic acid or lipoamide. The above observations suggested that P3 might possibly be replaced by lipoyl dehydrogenases. As shown in Fig. 8, both the pig heart and E. coli enzymes are able to substitute of P3 in the glycine oxidation system (Eq. 1). In this system, Pa exhibited higher activity than the pig heart enzyme in contrast to the results (cf. Fig. 3) obtained when the lipoyl dehydrogenase activity of these enzymes was compared. The pig heart enzyme can also replace P, when reversed electron flow (Eq. 3) is measured. The E. coli enzyme, however, was not active in this system, probably due to its inhibition by DPNH (13). As a corollary to this experiment, an attempt was made to see whether Pg could be replaced by lipoamide. In the overall reaction, however, Iipoamide was not able to
HUENNEKENS
replace P2, and in fact inhibited the complete system with P, present. These experiments further confirm the oxido-reduction function of P2 and P, in the overall reaction (Eq. 1) leading to the decarboxylation of glycine. They also establish the order with which these components carry the reducing power from glycine to DPN and provide an alternate method fur studying the electron transport portion of the overall system, namely the reversed electron flow between DPNH and an artificial acceptor, DTNB. Until PZ and Pa are obtained in highly purified form, it is not possible to draw any further comparisons between these proteins and the thioredoxinthioredoxin reductase system of Reichard and his colleagues (4, 5). PZ and Pa appear to be functionally similar to the enzymes of Reichard’s system, except for their specificity for DPN rather than TPN. As noted in our earlier paper (3), thioredoxin and thioredoxin reductase from E. coli will not substitute for PZ and Ps in the present system. Present evidence, especially the inhibitor
Flavin, mpmoles FIG. 8. Comparison of Pa, pig heart lipoyl dehydrogenase, and E. coli lipoyl dehydrogenase in the glycine oxidation system. The assay mixtures contained in 1.0 ml: 109 pmoles of phosphate buffer, pIi 7.0; 4 amoles of glycine, 1 Mmole of tetrahydrofolate; 25 rmoles of mercaptoethanol, 75 nqumoles of pyridoxal phosphate; 0.8 amole of AP-DPN; 0.32 mg of PI; 0.15 mg of Pz; 0.15 mg of PA; and varying amounts of Ps or lipoyl dehydrogenases as indicated. Enzymes were compared on the basis of flavin content (see legend for Fig. 3).
OXIDATIVE
DECARBOXYLATION
studies, supports our earlier contention that P2 has a reducible disulfide bridge. Moreover, the lipoyl dehydrogenase activity of P, is consistent with the view that the functional group of P, resembles lipoic acid. Authentic lipoyl dehydrogenases can replace P, in the overall glycine oxidation system, but it remains to be seen whether Pa is in fact a lipoyl dehydrogenase that functions with Pz as an alternate substrate or whether it is a reductase for Pz that can utilize lipoic acid or its amide as secondary substrates. ACKNOWLEDGMENTS The authors are indebted to Drs. Y. Hatefi and S. A. Kumar for helpful advice and suggestions during the course of this work, to Dr. U. J. Lewis for performing the electrophoretic analyses on acrylamide gel, and to Dr. Charles H. Williams for sending a sample of highly purified E. coli lipoyl dehydrogenase. REFERENCES 1. SAGERS, R. D., AND GUNSALUS, I. C., J. Bacteriol. 81, 541 (1961). 2. SAGERS, R. D., AND KLEIN, S. M., Federation Proc. 24, 219 (1965).
OF GLYCINE
711
3. BAGINSKY, M. L., AND HUENNEKENS, F. M., Biochem. Biophys. Res. Commun. 23, 666 (1966). 4. LAURENT, T. C., MOORE, E. C., AND REICHARD, P., J. Biol. Chem. 239, 3436 (1964). 5. MOORE, E. C., REICHARD, P., AND THELANDER, L., J. Biol. Chem. 239, 3445 (1964). in Enzymology” 6. LEVIN, O., in “Methods (8. P. Colowick and N. 0. Kaplan, eds). Vol. 5, p. 28. Academic Press, New York (1962). C. J., AND 7. GORNALL, A. G., BARDAWILL, DAVID, M. M., J. BioZ. Chem. 177, 751 f 1949). 8. KLEIN, S. M., AND SAGERS, R. D., J. BioZ. Chem. 241, 197 (1966). 9. KLEIN, S. M., AND SAGERS, R. D., Bacterial. Proc. p. 103 (1966). 10. ELLMAN, G. L., Arch. Biochem. Biophys. 83, 70 (1959). 11. MASSEY, V., GIBSON, Q. H., AND VEEGER, C., Biochem. J. 77, 341 (1960). 12. MASSEY, V., AND WILLIAMS, C. H., JR., J. BioZ. Chem. 240, 4447 (1965). 13. WILLIAMS, C. H., JR., J. BioZ. Chem. 240, 4793 (1965). 14. MASSEY, V., AND VEEGER, C., Biochem. Biophys. Acta. 48, 33 (1961).
OF
Further
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
on the Electron Oxidative
1”ARIETTA Department
of Biochemistry,
703-711 (1967)
ii?&
Transport
Decarboxylation L. BAGINSKY
Scripps
Clinic
Received
AND
Proteins
in the
of Glycine’ F. R/I. HUENNEKENS
and Research Foundation, December
Involved
La Jolla,
Cali)ornia
92037
20, 1966
The oxidative decarboxylation of glycine is catalyzed by a system consisting of 4 proteins isolated from Peptococcus glycinophilus. Ps, a small molecular weight, dithiolcontaining protein, and Ps, a flavoprot’ein, are concerned with electron transfer from glycine to DPN (i.e., glycine -+ Pz + P1 + DPN). Reversed electron flow through this sequence is also demonstrable (DPNH --) PI + Pp -+ DTNB). From its ability to be reduced by either DPNH or dihydrolipoic acid, and changes in absorption spectrum when the reduced enzyme is treated with arsenite or mercurials, PI appears to be a lipoyl dehydrogenase. Consistent with this hypothesis, lipoyl dehydrogenases from pig heart and Escharichia coli can replace Pa in each of the above transfer sequences.
Reversibility of this electron transport sequence was demonstrated by coupling the oxidation of DPNH to the chromogenic acceptor, 5,5’-dithiobis(2-nitrobenzoic acid), according to Eq. (3):
Sagers and Gunsalus (1) first demonstrated that, cell-free extracts of Peptococcus glllcinophilus were able to catalyze the oxidative decarboxylation of glycine according to the interesting stoichiometry of Eq. (1) : Glycine + tetrahydrofolate + DPN+ 4 5, lo-methylene tetrahydrofolate + CO, + NHd+ + DPNH.
DPNH + P3 ---f PZ + DTNB.2 (1)
Studies by Klein and Sagers (2) and by the present authors (3) have demonstrated that the enzyme system responsible for the above reaction can be resolved into four components: PI, a pyridoxal phosphate-containing protein; Pe, a heat-stable protein; PD, a flavoprotein; and P4, an uncharacterized protein. In a recent communication (3) we have proposed a mechanism for reaction (1) in which oxidation at the a-carbon of glycine is linked to DPN via fractions P2 and Ps, as shown schematically in Eq. (2) : Glycine + PZ -+ P3 + DPN.
(2)
1 Paper IX in the series “Flavin Nucleotides and Flavoproteins.” This work was supported by grants from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health (AM 08398), and the American Heart Association (63-G-146 and 66-796).
(3)
PZ is a low molecular weight, heat-stable protein whose properties and function closely resemble those of thioredoxin, an oxido-reduction component of the ribonucleotide reductase system in Escherichia coli (4). Thioredoxin contains a disulfide group that is reduced to a vicinal dithiol by a TPNHdependent flavoprotein (5). Since P, likewise undergoes reversible oxidation-reduction in the systems represented by Eq. (2) and (3), we have previously proposed that P, also has a functional disulfide group that is reduced by Pa, a DPNH-dependent flavoprotein (3). The present communication provides further evidence in support of the roles of P? and P3 as oxido-reduction carriers intervening between glycine and DPN (reaction 1). Inhibitor studies are consistent with the presence of a functional disulfide bridge in Pt. P3 has been shown to possess many of the characteristics of a lipoyl dehydrogenase; conversely, lipoyl dehydrogenases from other
703
704 sources can replace overall reaction.
BAGINSKY
P, as a component
AND
in
the
IIUE?U’NEKENS
amide, or by redllction of AP-DPN (increase in absorbancy at 363 mp) with dihydrolipoic acid. In addition to t.he substrate and ppridine nucleoEXPERIMENTAL PROCEDURE tide, incubat,ion mist.ures co11taiued ph0sphat.e buffer, EI)TA, bovine serum albllmill, and cataMale&&. The following chemicals were oblytic amounts of the flavoprotein. tained from commercial sources: DPN, DPNH, In all of the above assays, substrates and et>pyridoxal phosphate, DTNB, dihydrolipoic acid, zymes were incubated for 2 minutes at 38” before lipaamide, and pig heart lipoyl dehydrogenase starting the reaction. When reversed electron flow (Sigma Chemical Co.) ; 3-acetylpyridine-DPN was studied, DTNB was added after the preincu(P. L. Biochemicals) ; N-ethylmaleimide (Sehwarz bation. Reactions were started bv adding the Bio-Research, Inc.) ; iodoacetamide (California pyridine nucleotide to t,he experimental cuvette. Corporation for Biomedical Research); and a The blank cuvette contained the same components standard solution of bovine serum albumin (Aras the experimental, except for the nucleotide. In mour Pharmaceutical Co.). Iodoacetamide was all cases, blanks without substrate were also run. recrystallized from methanol-water before use. Absorbancy changes were followed at 15-second A highly purified sample of lipoyl dehydrogenintervals for periods up to 4 minutes in a Beckase from E. coli was generously provided by Dr. man Spectrophotometer, model DU, at 38”. The C. H. Williams, University of Michigan. Tetrahyexact composition of the assay mixtures is given drofolate was a gift from Dr. V. S. Gupta of this in the legends of Tables and Figures. department. Hydroxylapatite gel was prepared Preparation of enzymes. Peptococcus glycinaccording to the procedure of Levin (6). ophilus was grown anaerobically in the medium Methods. Absorption spectra were determined of Sagers and Gunsalus (I), modified as described at room temperature (22-25”) with a Beckman previously (3). Protein fractions (PI, P,, Ps, and DK-2 recording spectrophotometer. Thunberg Pa) were separated from each other and purified cuvettes with the side arm replaced by a rubber by the procedure summarized in a previous comcap were used in the anaerobic experiments. The munication (3). The following is a typical prepacuvettes were evacuated and flushed with preration: Fifty-five gm of frozen cell-paste was purified Nz six to ten times; additions were made thawed and suspended in 165 ml of 0.02 M potasby injecting solutions through the rubber cap. sium phosphate buffer, pH 7.0, containing 0.01% Protein was determined by the biuret method of Na2S. Fift.y-ml aliqnots of this cell suspension Gornall et al. (7) with bovine serum albumin as were sonicated for 20 minutes in a Raytheon 10 standard. KC sonic oscillator cooled with circulat,ing wat,er Assay systems. For the determination of reacat -5”. Intact cells and debris were removed by tion (I) reduction of DPN (or AP-DPN)2 was folcentrifugation for 30 minutes at 17,000 rpm in the lowed spectrophotometrically at 340 rnp (or 363 SS-34 rotor of a Servdl RC-2 centrifuge. (Unless mF) in a system containing glycine, phosphate otherwise stated, all subsequent centrifugations buffer, pyridoxal phosphate, tetrahydrofolate were performed in the same manner.) The superplus mercaptoethanol, catalytic amounts of all natant fluid (182 ml, 24.8 mg protein/ml) was four enzymes, and DPN (or AP-DPN). Reversed kept frozen overnight. Protamine sulfate (22.6 electron flow (reaction 3) was studied by following the reduction of DTNB at 412 w in a system ml of a 2% solution) was added dropwise and the containing phosphate buffer, EDTA, catalytic pH of the mixture was adjusted to pH 6.7 by t)he addition of 1 N KOH. After the preparation had amounts of PZ and PI, and DPNH. DTNB solubeen immersed in an ice-bath for 20 minutes, t,he tions were prepared just prior to use by dissolving 10 #moles of DTNB in 1 ml of ethanol and dilut,ing precipit,ated nucleic acids were removed by cento a volume of 10 ml with 0.5 M Tris-chloride trifugation for 30 minutes. To the supernatant, (200 ml) 467 ml of ammonium sulfate buffer, pH 8.0. Two-tenths ml of this solution was solution used for assay systems in which the total volume solution saturated at 5” (pH 7) was slowly added. was 1.0 ml. After standing at 2-5” for 30 minutes, the precipiLipoyl dehydrogenase activity was determined tate (@70y0 ammonium sulfate fraction) was disby measuring either DPNH oxidation (decrease carded. Solid ammonium sll1fat.e (120 gm) was in absorbancy at 310 rnp) in the presence of liposlowly added to the supernatant fluid (467 ml) to obtain a satllrated solution. The solution was z Abbreviations used: DTNB, 5,5’-dithiobisstirred for one hour at 2-5’ and then centrifuged (2-nitrobenzoic acid) ; AP-DPN, 3.acetyl-pyridine for 45 minutes. The precipitat,e was dissolved in analogue of DPN; NEM, Nethylmaleimide; IAA, 0.02 M potassium phosphate buffer, pH 7.0 (7(r iodoacetamide; PCMS, p-chloromercuriphenyl 100% ammonium sulfate fraction) sulfonate. The above solution (20 ml) was chromato-
OXIDATIVE
DECAR~OXYLATION
graphed on a 4.5 X 42-cm column of Sephadex G-50 (fine) equilibrated with 0.02 M potassium phosphate buffer, pH 7.0. Elution was carried out with the same buffer at a rate of 12 drops per minute; 5-ml fractions were collected. The first protein fraction eluted was yellow-green, and this was followed by a yellow-brown fraction. Rubredoxin, ferredoxin, and free flavin remained on the column. Those tubes containing the yellow-brown fraction plus the last tube of the yellow-green fraction were pooled and lyophilized. This sample contained the bulk of the Pp activity. Those tubes containing the yellow-green fraction were pooled and concer,trated by ultrafiltration. The latter fraction, which contained P,, Pa, and P*, along with some Ps, could be stored frozen for 2 weeks without apparent loss in activity. For further purification of PZ, the lyophilized fraction was dissolved in 20 nil of water and heated for 3 -minutes in a boiling water bath. The sample was immediately cooled in an ice-bath and the precipitate was removed by centrifugation for 20 minutes at 40,000 rpm (No. 40 rotor) in a Spinco ultracentrifuge. The supernatant solution was dialyzed for 6 hours against 4 liters of 0.02 M potassium phosphate buffer, pH 6.8 (one change of buffer), and concentrated by the use of dry Sephadex G-25. Two such fractions were pooled (32 ml, 5 mg protein/ml) and chromatographed on a 2.8 X 4:1-cm column of hydroxylapatite that had been equilibrated with 0.02 M potassium phosphate buffer: pH 6.8. Gradient elution was performed with potassium phosphate buffer, pH 6.8 10.05 -+ 0.2 M, 800 ml each). The flow rate was 11 drops per minute and fractions of 80 drops were collected. Two principal protein peaks3 were obtained: a salmon-colored fraction in tubes 68-85 and a colorless fraction (slightly yellow in very concentrated solutions) in tubes 90-120. PZ activity was found in both peaks, but the specific activity of the colorIess fraction was four times that of the other. Tubes 90-120 were pooled, concentrated with dry Sephadex G-25, and kept frozen until use. For the separation of P1, I’s, and P4, the concent.rated yellow-green fraction described above (50 ml, 25 mg prot.ein/ml), was chromatographed on a 3.5 X 4O-cm column of D~AE-cellulose that had been equilibrated with 0.02 M potassium phosphate buffer, pH 7.0. Gradient elution was performed with potassium phosphate buffer, pH 7.0 (0.05 --t 0.4 M, 800 ml each). Five-ml fractions were collected at a rate of 15 drops per minut,e. The first peak contained the Pt activity, t,he see3 Klein and Sagers (8) have previously reported the separation of Pz into two peaks of activity by chromatography on the same adsorbant.
706
OF GLYCINE
ond peak was discarded, and P1 and P) were found in the third and fourth pea.ks, respectively. Solutions containing each of these activities were pooled, concentrated by ultrafiltration, and kept frozen. Each of the above three fractions was purified further by chromatography on hydroxylapatite. A 3.5 X 20-cm column, equilibrated with 0.02 M potassium phosphate buffer, pH 6.8, was used for 150 mg of each fraction. Elution was performed with a linear gradient of potassium phosphate buffer, pH 6.8 (0.05 + 0.3 M, 800 ml each). The flow rate was 12 drops per minute. Solutions containing the desired activities were pooled, concentrated by ultrafiltration, and stored frozen. Pt was further purified by ammonium sulfate precipitation (70-77% fraction). This fract,ion was dissolved in 0.05 M potassium phosphate buffer, pH 7.2, and used directly, or desalted by passage through Sephadex G-25. At this stage of purification each of the four proteins was not contaminated with any of the other three. Pq gave a single band when examined by electrophoresis on acrylamide gel, but each of t’he other fractions displayed, in addition to the principal band, several other weak contaminating bands. A somewhat different procedure for the purification of Pt and Pz has been used by Klein and Sagers (S), and recently, the same authors have described an excellent new procedure for the separation of the four proteins (9). RESULTS
AND
~eq~~~~~~.~n~ for
DISCUSSION
the olierall
r~~et~~.
As
shown by Klein and Sagers (9) and also by TABLE REDUCTION
OF DTNB
I BY GLYCINE
Componenl.omitted None” Glycine Tetrahydrofolate Pt P*
21.2 0.3 0.3 0.3 3.8
aThe complete system contained in 1.0 ml: lOOpmoles of phosphate buffer, pH 7.0; 75 mpmoles of pyridoxal phosphate; 2 pmoles of tetrahydrofolate; 4 rmoles of glycine; 0.2 #mole of DTNB (in ethanol-Tris buffer as described in the Experimental Section); 0.8 mg of PI; 0.25 mg of Pp; and 0.4 mg of Pg. Blank cuvettes contained all components except Pz. b Based upon E = 13.6 X lo3 M-’ cm-’ at 412 ml* (lo).
BAGINSKY
706 TABLE DPNH-DEPENDENT Component omitted
None0 PZ PP DPNH
AND
II
REDUCTION
OF DTNB DTNB reduced (mpmoles/min)
10.6 0.01 0.03 0.00
a The complete system contained in 1.0 ml: 50 pmoles phosphate buffer, pH 7.2; 10 pmoles of EDTA; 0.2 pmole of DTNB (in ethanol-Tris buffer); 0.3 rmole of DPNH; 0.03 mg of P,, and 0.01 mg of Pa. For the first three experiments, the blank cuvettes contained all components except DPNH. When DPNH was the component omitted, P2 was deleted from the blank.
our studies (3), the oxidative decarboxylation of glycine linked to DPN (Eq. I), requires four protein fractions (PI, Pg, Pa, and P4) in addition to tetrahydrofolate and pyridoxal phosphate as cofactors. The requirement for tetrahydrofolate is absolute, while that for pyridoxal phosphate is partial and variable depending upon the extent to which PI is present as the apoenzyme. E~ta~lishe~nt of Pz and P3 as orido-yeduetion carriers between ylycine and DPN. In the mechanism that we have presented previously (3), it was postulated that oxidation and decarboxylation of glycine occur as a concerted reaction, and that the reducing power generated in this step is carried to DPN via Pt and Pt (cf. Eq. 2). Support for the placement of P, prior to P3 in the electron flow from glycine to DPN was provided by the observation, illustrated as Fig. 1 in our previous paper (3), that dithionite-reduced P8 is rapidly oxidized by DPK, but not by TPN; this specificity for pyridine nucleotide is in accordance with that noted for the overall reaction. In addition, oxidation of glycine can occur in the absence of P3 and DPN provided that an external electron acceptor, such as DTNB, is present (Table I). All of the other components of the complete system (Eq. 1) are required for the DTNB-linked oxidation of glycine. Although not indicated in Table I, omission of PZ completely abolished the reduction of DTNB; mixtures without Ps could be used, therefore, as blanks.
HUENNEKENS
Reversed electron $ow. It was also convenient to study the function and relation ship of P, and P, in the reverse sequence, i.e., by generating the reducing power from DPNH, rather than glycine. As described by Eq. (3), oxidation of DPNH can be coupled to the reduction of DTNB in the presence of catalytic amounts of Pz and Ps (Table II). This experiment also provided evidence that each of these proteins was free from contamination by the other. As predicted by the sequence of Eq. (3), DPNH can be used to reduce substrate amounts of PB (Fig. 1). The absorption band of P, in the 400-500 rng region, characteristic of oxidized flavoproteins, was replaced by a lower and more broad band at a somewhat lower wavelength when progressively larger amounts of DPNH were added. At the same time the absorbance between ,500 and 700 mp increased, probably due to formation of a charge-transfer complex between the flavin semiquinone and DPN. This point will be discussed further in a later section of the paper. Under these conditions, the solution in the cuvette changed from yellow to red. Similar results have been reported for other flavoproteins reduced by their substrates (11, 12). Complete reduction of Pa, as indi-
300
400 500 600 Wavelength,mp
700
FIG. 1. Reduction of Pa by DPNH. The experimental cuvette contained in a final volume of 1.0 ml: 5 mg of P1; 70 pmoles of phosphate buffer, pH 7.3; 2 rmoles of EDTA; and 40 pmoles of ammonium sulfate. Oxidized Pa (p); plus 0.01 ml of 10 mu DPNH (- - -); plus an additional 0.03 ml of DPNH (. . . . ); opened to air plus addition of dithionite (-.-.). For the first two spectra the blank cuvette contained water; 1 ml water plus 0.02 ml of 10 mM DPNH was used for the blank in the latter two spectra. Additions of DPNH were made under anaerobic conditions.
OXIDATIVE
DECARBOXYLATION
I
300 400 500 600 70~? Wavelength,mp FIG. 2. Oxidation of reduced Pa by Pt. The experimental cuvette contained in a final volume of 1.0 ml: 5 mg of P3; 70 pmoles of phosphate buffer, pH 7.3; and 2 Mmoles of EDTA. Oxidized Pa (+-) ; reduced with dithionite (under N2) (-.-. ) ; plus 0.02 ml of PZ (17.7 mg protein/ml) (. . . .); plus an additional 0.02 ml of P, (- - -). Water was used as the blank for the first three spectra while 1.0 ml of water plus 0.04 ml of PS was used for the final spectrum. Additions of P2 were made under anaerobic conditions.
cated by abolition of the 450 and 550 rnp bands, was obtained by the addition of dithionite. Again, from Eq. (3), it should be possible to oxidize reduced P3 with substrate amounts of P,. This is demonstrated in Fig. 2. When P, was fully reduced with dithionite and then treated with successively larger amounts of oxidized Pz, the absorbancy at 455 rnp returned to a value close to that of the fully oxidized enzyme, while that in the 500-600 rnp region was higher than the original absorbancy. In this experiment the P2 used was assumed t’o be in the oxidized for+ as isolated; this assumption is supported by observations described below. Finally, oxidation of DPNH can be linked to PZ, provided that PB is present in catalytic amounts. The experiments described in the next section make use of this reaction to prepare substrate amounts of reduced Pz. Evidence that Pp contains a functional dithiol group. The possibility that the oxidoreduction component of PZ is a vicinal dithiol group, sitiilar to that occurring in thioredoxin (4), was investigated with the use of sulfhydryl inhibitors. As shown in Table III, iodoacetamide and NEM inhibited the reduced form. of Pz but not the oxidized form.
707
OF GLYCINE
In these experiments P, was reduced enzymically (by incubation under anaerobic conditions with DPNH and P3) and then incubated with iodoacetamide or NEM under anaerobic conditions. Exce& inhibitor was removed4 by passage of the mixture through Sephadex G-25. A control sample was treated in the same manner except that water was added instead of the inhibitor. Activity of P, was then determined by measuring DTNB reduction in the presence of additional PB and DPNH. Experiment 1 in Table III shows that samples of P, treated with either iodoacetamide or NEM were almost inactive when compared with the untreated control. That the observed effect was not caused by inhibition of P, rather than PZ, due to inTABLE INHIBITION
OF AND
Experimalt NO.
REDUCED
III Pz
BY
IODOACETAMIDE
N-ETHYLMALEIMIDE~
Sample
DTNB reduced (U moles/ min)
Ps used Specific in assay activity* bw)
1
Reduced Reduced Reduced
PZ Pp + IAA Pz + NEM
28.1 0.66 0.66
0.082 0.078 0.079
329 8 8
2
Reduced Oxidized Reduced
Pz Pz + NEM P, + NEM
31.7 40.6 0.96
0.090 0.089 0.090
338 436 1
u Under anaerobic conditions Pt (3.54 mg) was reduced by treatment with 2.5 pmoles of DPNH, 0.15 mg of Pz, and 50 Mmoles of potassium phosphate, pH 7.2 (total volume, 0.65 ml) for 15 minutes at 38”, and then incubated for 60 minutes at 38” wit,h either 10 mM iodoacetamide or 4 mM NEM. The preparation was then passed through a 1 X 20.cm column of Sephadex G-25 that had been equilibrated with 0.05 M phosphate buffer, pH 7.0; elution was accomplished with the same buffer. An aliquot of P, was assayed for activity in a system that contained in 1.0 ml: 50 pmoles of phosphate buffer, pH 7.2; 10 rmoles of EDTA; 0.2 rmole of DTNB; 0.3 pmole of DPNH; 0.044 mg of PI; and amounts of treated P? as indicated. * mpmoles DTNB reduced/min/mg protein. 4 Addition of the inhibitors directly to the assay system (DPNH + Pt --t Pp + DTNB) would have not revealed which of the components had been inactivated.
705
BAGlNSKY
0.01
0.02
0.03
0.04
AND
0.05
Flaw, mpmoles FIG. 3. Lipoyl dehydrogenase activity of Ps. Assay mixtures contained in 1.0 ml: 60 pmoles of phosphate buffer, pH 7.3; 2 pmoles of EDTA; 2.4 pmoles of dihydrolipoic acid; 0.6 mg of bovine serum albumin; 0.8 /*mole of AP-DPN; and varying amounts of the enzymes as indicated. Concentration of each enzyme is expressed in terms of flavin content. Flavin was determined by measuring the decrease in absorbancy at 455 rnp following addition of dithionite and using a differential extinction coefficient of 11.3 X lo3 MM' cm-l.
complete removal of the inhibitor, was borne out by Experiment 2. When both t,he oxidized and reduced forms of Pz were incubated with NEM, only the latter was inhibited. Repetition of this experiment several times showed that treatment of the oxidized form with NEM occasionally produced a stimulation of the activit,y. When the above experiment was repeated with 10 mM Cd++ or arsenite as inhibitors (at pH 5.0 to prevent precipitation of Cd++), the results were less conclusive. In both cases the observed inhibition was only 3040 %. The failure of these reagents to completely inhibit Pz, as would be expected for a vicinal dithiol, may be due to dissociation of the enzyme-inhibitor complexes upon passage through Sephadex.5 Relationship of Pa to lipoyl clehydrogenases. The absorption spectrum of P, in the visible region is very similar to t,hat of a lipoyl dehydrogenase (cf. Fig. 2 of Ref. 13). It was not surprising, therefore, to find that P3 5 Arsenite (1W M) and CdCl, (lo* M), when added directly to the assay system, inhibited the overall reaction (Eq. 1) by 72 and 739&, respectively.
IIUENNEKENS
exhibited good lipoyl dehydrogenase activity lvhen tested eit,her by reduction of Al’-DPN with dihydrolipoic acid or by reduct.ion of lipoamide with DPJSH. The data of Fig. 3 compare the ability of Pa, pig heart lipoyl dehydrogenase, and E’. coli lipoyl dehydrogenase to catalyze the reduction of AP-DPN by dihydrolipoic acid; each enzyme was tested at several levels based on flavin eoncentration. Although the pig heart’ enzyme exhibited the greatest activity, Pa was also an effective lipoyl dehydrogenase. Similarly, the pig heart enzyme and P, showed the same activity in catalyzing the DPNHdependent reduction of lipoamide. Lipoyl dehydrogenases are known to be inhibited by DPNH but not by AP-DPNH. Since Ps, when tested as a lipoyl dehydrogenase, n-as also sensitive to DPNH, it seemed likely that our earlier data, in which the overall reaction (Eq. 1) was characterized by a decline in rate with t,ime, would have been consistent with inhibition by one of the products, DPNH. As shown in Fig. 4, AP-DPN not only replaces DPN in the
Time, min. FIG. 4. Comparison of DPN and AP-DPN in the glycine oxidation system. The assay mixtures contained in a volume of 1.0 ml: 75 pmoles of phosphate buffer, pH 7.0; 1 pmole of tetrahydrofolate; 25 pmoles of mercaptoethanol; 75 mpmoles of pyridoxal phosphate; 4 pmoles of glycine; 0.35 mg of protein (PI + Pz + Pa + P4) and 0.8 pmole of DPN or AP-DPN.
OXIDATIVE
DECARBOXYLATION
Wavelength,mp FIG. 5. Reduction of PB by dihydrolipoic acid. The experimental cuvette contained initially the same components as described in the legend for Fig. 1. Oxidized Pa (---); plus 0.01 ml of 12 mM dihydrolipoic acid (- - -) ; plus an additional 0.93 ml of dihydrolipoic acid (. .); opened to air, plus dithieonite (-.-.). Additions of dihydrolipoic acid were made under anaerobic condit.ions.
overall assay system but also abolishes the decline in activity seen with the former. It was shown previously (l;ig. 1) that DPNH can reduce I’$ to a red, presumably scmiquinone, stage. The same result was obtained when P)3was reduced anaerobically Lvith dih,ydrolipoic acid (Fig. 5). Even upon addition of a large excess of dihydrolipoic acid (0.96 pmole per 0.0‘25 pmole of flavin) the enzyme \vas not reduced beyond the scmiquinone form; complete redu&ion was obtained, however, with dithionite. In agreement with the findings of Massey and Veeger (14)) almost identical results were obtained with the pig heart enzyme. In contrast, the E. co&i enzyme gradually assumes the fully reduced form as the concentratjion of substrate is increased (13). The above findings prompted us to test P, for other characteristics that have been reported for pig heart and E. coli lipoyl dehydrogenases and yeast glut#athione reductase. Massey and his co-workers (11-14) have proposed that the above enzymes contain a disulfide bridge which, upon reduction, permit
the
transfer
of one electron
from
one of
the sulfhydryl groups to bhe flavin; interaction of the resultant sulfur radical with the flavin semiquinone would stabilize both groups. This proposed complex is characterized by an absorption maximum at about 530 rnp, and its interaction with DPNH is
709
OF GLYCINE
believed to form a charge-transfer complex with absorption maximum at about 570 rnp. Formation of the latter explains the broader absorption band above 500 rnp observed when the above enzymes are reduced with DPNH. The same results have also been obtained with P3 (cf. Figs. 1 and 2). Also, as found by the above authors, addition of PCMS to the DPNH-reduced enzymes resulted in complete reduction of the flavin; the same result is observed with DPNHreduced P3 (Fig. 6). Finally, as reported for the lipoyl dehydrogenases and the yeast glutathione reductase, addition of increasing amounts of DPNH to the arsenite-treated enzyme resulted in progressive bleaching of the 455 rnp band accompanied by formation of a green band with absorption maximum at 720 rn#. The latter has been attributed to the formation of a charge-transfer complex between the fully reduced enzyme and DPlS. A
400
500
600
700
800
Wavelength, mp
FIG. 6. Effect of PCMS on DPNH-reduced Ps. The experimental cuvette contained in a total volume of 1.0 ml: 80 pmoles of phosphate buffer, pH 7.3; 2 rmoles of EDTA; and 11.9 mg of Pa. Oxidized PB (blank: water) (--) ; plus 0.02 ml of 20 rnM DPNH (blank: 1 ml water plus 0.01 ml of DPNH) (- - -); plus an additional 0.02 ml of DPNH 1 hour later (blank: plus an additional 0.01 ml of DPNH) (. .); plus 0.02 ml of 50 mM PCMS added to both experimental and blank cuvettes (-.-.). Additions of DPNH and PCMS were made under anaerobic conditions.
710
BAGINSKY
AND
Wavelength,q FIG. 7. Reduction of arsenite-treated Pa by DPNH. The experimental cuvette contained in a total volume of 1.0 ml: 80 pmoles of phosphate buffer, pH 7.3; 2 Gmoles of EDTA; 1 pmole of sodium arsenite and 9.85 mg of PI. The blank cuvette contained the same components with the exception of the enzyme. Oxidized Ps (--); plus 0.01 ml of ‘20 mM DPNH (blank: plus 0.004 ml of DPNH) (- - -); plus an additional 0.01 ml of DPNH (blank: plus an additional 0.004 ml of DPNH) (-.-.); plus an additional 0.02 ml of DPNH (blank: plus an additional 0.01 ml of DPNH) (...-). Additions of DPNH were made under anaerobic conditions.
comparable experiment, involving Pa, is shown in Fig. 7. Possible replacement of P3 by lipoyl dehydrogenase and of P9 by dihydrolipoic acid or lipoamide. The above observations suggested that P3 might possibly be replaced by lipoyl dehydrogenases. As shown in Fig. 8, both the pig heart and E. coli enzymes are able to substitute of P3 in the glycine oxidation system (Eq. 1). In this system, Pa exhibited higher activity than the pig heart enzyme in contrast to the results (cf. Fig. 3) obtained when the lipoyl dehydrogenase activity of these enzymes was compared. The pig heart enzyme can also replace P, when reversed electron flow (Eq. 3) is measured. The E. coli enzyme, however, was not active in this system, probably due to its inhibition by DPNH (13). As a corollary to this experiment, an attempt was made to see whether Pg could be replaced by lipoamide. In the overall reaction, however, Iipoamide was not able to
HUENNEKENS
replace P2, and in fact inhibited the complete system with P, present. These experiments further confirm the oxido-reduction function of P2 and P, in the overall reaction (Eq. 1) leading to the decarboxylation of glycine. They also establish the order with which these components carry the reducing power from glycine to DPN and provide an alternate method fur studying the electron transport portion of the overall system, namely the reversed electron flow between DPNH and an artificial acceptor, DTNB. Until PZ and Pa are obtained in highly purified form, it is not possible to draw any further comparisons between these proteins and the thioredoxinthioredoxin reductase system of Reichard and his colleagues (4, 5). PZ and Pa appear to be functionally similar to the enzymes of Reichard’s system, except for their specificity for DPN rather than TPN. As noted in our earlier paper (3), thioredoxin and thioredoxin reductase from E. coli will not substitute for PZ and Ps in the present system. Present evidence, especially the inhibitor
Flavin, mpmoles FIG. 8. Comparison of Pa, pig heart lipoyl dehydrogenase, and E. coli lipoyl dehydrogenase in the glycine oxidation system. The assay mixtures contained in 1.0 ml: 109 pmoles of phosphate buffer, pIi 7.0; 4 amoles of glycine, 1 Mmole of tetrahydrofolate; 25 rmoles of mercaptoethanol, 75 nqumoles of pyridoxal phosphate; 0.8 amole of AP-DPN; 0.32 mg of PI; 0.15 mg of Pz; 0.15 mg of PA; and varying amounts of Ps or lipoyl dehydrogenases as indicated. Enzymes were compared on the basis of flavin content (see legend for Fig. 3).
OXIDATIVE
DECARBOXYLATION
studies, supports our earlier contention that P2 has a reducible disulfide bridge. Moreover, the lipoyl dehydrogenase activity of P, is consistent with the view that the functional group of P, resembles lipoic acid. Authentic lipoyl dehydrogenases can replace P, in the overall glycine oxidation system, but it remains to be seen whether Pa is in fact a lipoyl dehydrogenase that functions with Pz as an alternate substrate or whether it is a reductase for Pz that can utilize lipoic acid or its amide as secondary substrates. ACKNOWLEDGMENTS The authors are indebted to Drs. Y. Hatefi and S. A. Kumar for helpful advice and suggestions during the course of this work, to Dr. U. J. Lewis for performing the electrophoretic analyses on acrylamide gel, and to Dr. Charles H. Williams for sending a sample of highly purified E. coli lipoyl dehydrogenase. REFERENCES 1. SAGERS, R. D., AND GUNSALUS, I. C., J. Bacteriol. 81, 541 (1961). 2. SAGERS, R. D., AND KLEIN, S. M., Federation Proc. 24, 219 (1965).
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3. BAGINSKY, M. L., AND HUENNEKENS, F. M., Biochem. Biophys. Res. Commun. 23, 666 (1966). 4. LAURENT, T. C., MOORE, E. C., AND REICHARD, P., J. Biol. Chem. 239, 3436 (1964). 5. MOORE, E. C., REICHARD, P., AND THELANDER, L., J. Biol. Chem. 239, 3445 (1964). in Enzymology” 6. LEVIN, O., in “Methods (8. P. Colowick and N. 0. Kaplan, eds). Vol. 5, p. 28. Academic Press, New York (1962). C. J., AND 7. GORNALL, A. G., BARDAWILL, DAVID, M. M., J. BioZ. Chem. 177, 751 f 1949). 8. KLEIN, S. M., AND SAGERS, R. D., J. BioZ. Chem. 241, 197 (1966). 9. KLEIN, S. M., AND SAGERS, R. D., Bacterial. Proc. p. 103 (1966). 10. ELLMAN, G. L., Arch. Biochem. Biophys. 83, 70 (1959). 11. MASSEY, V., GIBSON, Q. H., AND VEEGER, C., Biochem. J. 77, 341 (1960). 12. MASSEY, V., AND WILLIAMS, C. H., JR., J. BioZ. Chem. 240, 4447 (1965). 13. WILLIAMS, C. H., JR., J. BioZ. Chem. 240, 4793 (1965). 14. MASSEY, V., AND VEEGER, C., Biochem. Biophys. Acta. 48, 33 (1961).