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Crystalline flavin pyruvate oxidase from Escherichia coli
ARCHIVES
OF BIOCHEMISTRY
Crystalline
AND
BIOPHYSICS
Flavin
Pyruvate
I. Isolation
B. Conant Laboratory, and the Biochemistry
168-176
WILLIAMS3
(1966)
Oxidase
and Properties
F. ROBERT James
116,
Escher-i&a
co/i
of the Flavoproteit? AND
LOWELL
Department of Chemistry, Harvard Division, Departwlent of Chemistry University of Illinois, Urbana, Received
from
May
P. HAGER4
University, Cambridge, Massachusetts and Chemical Engineering, Illinois
16, 1966
Pyruvate oxidase has been isolated from Escherichia coli in crystalline form and at, a high state of purity. Pyruvate oxidase binds both flavin-adenine dinucleotide and thiamine pyrophosphate as prosthetic groups. Molecular weight measurements indicate that the crystalline oxidase exists in solution as a tetramer. The minimal molecular weight of the oxidase, based on Aavin-adenine dinucleotide content, is 66,060, whereas the molecular weight of the oxidase, as measured by equilibrium sedimentation, is approximately 265,000. This paper reports the spectral properties, the amino acid content, the kinetic constants, and some of the enzymic activities of crystalline oxidase.
Previous communications (l-5) from this laboratory have reported the presence of a flavin pyruvate oxidase system in Escherichia coli. This enzyme system catalyzes the oxidative decarboxylation of pyruvate to acetate and carbon dioxide according to Eq. (1): CH,COCOOH
+ 36 02
TPP,Mg++, terminal
As indicated in Eq. (I), TPP,5 Ng++, and a cytochrome-containing terminal electron transport system are required (1, 4, .5) in addition to the flavoprotein for catalysis in this reaction. The terminal electron system of E. coli can be obtained in the form of a particulate fraction which sediments at high gravitational forces (4). This flavoproteincytochrome system for pyruvate oxidation is distinct and separate from t’he CoA, DPNf, and lipoic acid-pyruvate dehydrogenase complex which is also present in E. coli (6, 7). Ferricyanide can replace oxygen as an electron acceptor in Eq. (1). In this case, the flavoprotein reacts sluggishly with ferricyanide in t’he absence of the cytochromecontaining particulate fraction. However, the rate of the ferricyanide-linked reaction can be increased approximately 25-fold by several means. Perincubation of the flavoprotein with trypsin (2), or the addition of certain surface-active agents, such as
electron
flavoprotein transport
plus
a
system CH&OOH
> + CO2
1 Dedicated to Luis F. Leloir on the occasion of his sixtieth birthday. 2 This investigation was supported by a grant (RG 7768) from the National Institutes of Health. Portions of this work were taken from a thesis submitted by F. Robert Williams in partial satisfaction of requirements for a degree of Doctor of Philosophy, Harvard University. A preliminary report of this work has appeared (1). 3 This author held a predoctoral fellowship from the National Institutes of Health at the time of investigation. Present address: Service de Biochimie, Centre d’etudes Nucleaires de SaclayGif-sur-Yvette, S. et 0. France. 4 Reprint requests should be addressed to this author at Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois.
6 Abbreviations used: DPN+, diphosphopyridine nucleotide; FAD, flavin-adenine dinucleotide; FMN, flavin mononucleotide; INT, 2-(piodophenyl)-3-p-nitrophenyld-phenyl tetrazolium chloride; TPP, thiamine pyrophosphate; TTC, triphenyl tetrazolium chloride. 168
CRYSTALLINE
PYRUVATE
sodium lauryl sulfate, or the addition of the particulate fraction (4) or solubilized preparations derived from t’he particulate fraction (5) markedly enhance the activity of the flavoprotein with ferricyanide. Acetone extracts of the particulate fraction also activate the flavoprotein in the ferricyanidelinked react,ion (3). We shall present in this paper the final purificat,ion and cryst’allization of the flavoprotein component of this system and report on some of the physical and catalytic propert’ies of the enzyme. M.$TERIALS
AND
METHODS
Enzyme assay. The flavoprotein is routinely assayed by measuring carbon dioxide release from pyruvate with the ferricyanide serving as the electron acceptor. The assay reaction mixture contains 1ClOrmoles of potassium phosphate buffer, pH 6.0; 0.1 Imole of TPP; 10 @moles of MgCl:; 50 pmoles of potassium pyruvate; 0.5 mg of ovalbumin; 1 pmole of sodium lauryl sulfate; 25 pmoles of potassium ferricyanide; and sufficient enzyme to catalyze the evolution of 0.3-3 pmoles of carbon dioxide per 5.minute interval. The reaction is started by t,he addition of ferricyanide from the sidearm of the Warburg cup. The incubation temperature is 31”. One unit of enzymic activity in the ferricyanide assay is defined as the evolution of 1 rmole of carbon dioxide per 30 minutes under the assay conditions described above. The rate of carbon dioxide evolution is estimated from the first 10 minutes of t.he reaction. Specific activity is defined as units per milligram of protein. Oxygen assay. The flavin pyruvate oxidase is nonautoxidizable in the form of its enzymesubstrate complex; however, oxygen uptake can be observed in a coupled system when the oxidase is supplemented with a particulate fraction derived from the cell envelope. This assay, which has been previously described (2), can be used both to estimate the act,ivity of flavoproteiu in the presence of an excess of the particulate fraction and the enzymic activity of the particulate fraction in the ‘presence of an excess of flavoprotein (4). Protein determinations. Protein concentrations were estimated by three different methods depending on the purity of the enzyme. If the ratio of 280 to 260 mp absorption was 0.6 or less, the protein concentrat:lon was measured by the trichloroacetic acid precipitation method (8). The protein concentrat[ons of fractions having a 280/260 ratio of 0.6 or more were measured by the WarburgChristian method (9). The concentration of crystalline enzyme was obtained from the absorption
169
OXIDASE
at 280 rn/* with a multiplication factor of 0.84 for the conversion of absorbance (1 cm lightpath) to milligram of protein per milliliter. The conversion factor was based on dry-weight det,erminations. ~1Z&riaZs. Commercial preparations of FAD, FMN, riboflavin, and TPP were obtained from the Sigma Chemical Corp. Potassium pyruvate, ovalbumin, and protamine sulfate were products of Nutritional Biochemicals Corp. DEAE-Sephades was obtained from Pharmacia Co. Flavin-adenine dinucleotide and flavin mononucleotide standards were prepared by ascending paper chromatography of commercial FAD and FMN; a solvent system composed of t-butanol and water (60:40) was used. The spots corresponding t,o FAD and FMN were cut out, eluted in the cold, checked by spectral analysis, and stored at 4” in the dark. All operations involving free flavins were carried out wherever possible at 4” in the dark.
Enzyme PuriJication Growth medizcm. The cells are grown on a medium containing 0.2% NH&l; 0.4% glucose; 0.25% sodium glutamate; 0.0005~o yeast extract; 0.150/, KH?PO*; 1.35% NazHPOa; 0.02% MgSOa.7 H,O; O.OOl’% CaC12; and 0.00005% FeSOI.7 H?O. The phosphate, glutamate, and glucose are sterilized in separate containers and added to the sterile salt solution. When the acetate mutant is used as the source of enzyme, the medium is further supplemented with 20 pmoles of potassium acetate per milliliter. The cells are grown under forced aeration, either in lo-liter batches in a New Brunswick fermentor, or in loo-liter batches in a 5C-gallon fermentor manufactured by Stainless and Steel Products Co. Inoculum. Escherichia coli, strain B, Crookes, W, or the acetate-requiring mutant 191-6 (7) derived from strain W, is grown on a complex medium containing 1% tryptone, 1% yeast extract, 0.59/ KZHPOI, and 0.3% glucose. Actively growing cells (log phase) are inoculated at a 0.5% level into the growth medium. Growth. Cells are grown to the stationary phase (15-20 hours) and are then harvested on a Sharples centrifuge. The yield is approximately 10 gm of cell paste per liter of culture medium. Preparation of cell extract. Six hundred gm of cell paste is suspended in 500 ml of 0.02 M potassium phosphate buffer, pH 7,6 and mixed thoroughly in a Servall omnimixer. Six hundred ml of washed glass beads (Minneapolis Mining and 0 Unless otherwise to buffer, phosphate buffer refer to 0.02 pH 7.
noted, subsequent references buffer, or standard phosphate M potassium phosphate buffer,
170
WILLIAMS
Manufacturing Co., Minnesota), 120 p in diameter, are added to the cell suspension. The cell-glass bead mixture is then ground in an Eppenbach colloid mill (rotor-stator setting of 0.030) for 30 minutes at 15-20” with continuous recycling. The resulting slurry is centrifuged at 15,OOOg for 10 minutes to remove the glass beads. The supernatant fluid (supernatant 1) is saved. The precipitate (together with the glass beads) is washed by resuspension in 1000 ml of phosphate buffer, and is then centrifuged at 15,000g for 10 minutes. The supernatant fluid (supernat,ant 2) is again saved. The precipitate (consisting mostly of glass beads) is again resuspended in 1000 ml of phosphate buffer and allowed to sediment for 1 hour. The supernatant fluid (supernatant 3) is decanted and saved. Supernatants 1 and 2 are combined and centrifuged at 15,000g for 1 hour. Three layers are formed during the centrifugation: a clear liquid phase; a dense, viscous particulate phase; and a precipitate of cell debris. The liquid phase and the particulate phase are removed toget,her and saved (supernatant 4). The precipitate of cell debris is washed by resuspension in supernatant 3 and is centrifuged at 15,000g for 1 hour. The precipitate is discarded. The supernatant fluid (supernatant 5) is removed and combined with supernatant 4 t’o yield a crude extract. First ammonium suljate precipitation. The crude extract is brought to 0.25 ammonium sulfate saturation by the addition of 14.4 gm of ammonium sulfate per 100 ml of crude extract. The suspension is stirred for 4 hours. The precipitate is removed by centrifugation at 15,OOOg for 75 minutes. This precipitate is saved and is used for the preparation of crystalline cytochrome bl (5). The supernatant fluid is brought to 0.75 ammonium sulfate saturation by the addition of 34.4 gm of ammonium sulfate per 100 ml of supernatant fluid. The precipitate is removed by centrifugation at 15,000g for 30 minutes and redissolved in a volume of buffer equal to one-half the volume of the initial crude extract. Removal of nucleic acid. The 0.25-0.75 saturated ammonium sulfate fraction is adjusted to pH 6 by the addition of 1 N acetic acid. Nucleic acid and inactive protein are precipitated by the slow addition of an equal volume of 2$!$ protamine sulfate, pH 6. The resulting mixture is dialyzed overnight against a total of 20 volumes of phosphate buffer. The insoluble protamine nucleate, which precipitates during dialysis, is removed by centrifugation at 15,000g for 20 minutes and discarded. At this stage the ratio of ultraviolet absorption at 280 rnp and 260 mp (280/260) of the supernatant solution should be above 1, although successful purifications have been carried out with fractions having a ratio of 0.8.
AND
HAGER
Second ammonium suljaie precipitation. The supernatant fluid from the protamine sulfate treatment is brought to 0.36 ammonium sulfate saturation by the addition of 25.2 gm of ammonium sulfate per 100 ml of supernatant fluid. After stirring for 2-3 hours, the resulting precipitate is removed by centrifugation at 15,000g for 30 minutes and discarded. The supernntant fluid obtained from the 0.36 saturated ammonium sulfate fraction is brought to 0.55 ammonium sulfate saturation by the addition of 12.1 gm of ammonium sulfate per 100 ml of supernntant fluid and stirred for 2-3 hours. The precipit,ate is removed by centrifugation at 15,OoOg for 30 minutes and redissolved in a volume of buffer equal to 0.06 volume of the crude extract. Heat precipitation. The 0.36-0.55 saturated ammonium sulfate fraction is rapidly warmed to 60’ in a 70-80” bath with vigorous stirring and then held at 60” in a 60” bath for 5 minutes. The heattreated fraction is rapidly cooled in an ice bath and the precipitate of denatured protein is removed by cent.rifugation at 35,000g for 1 hour. The precipitate of denatured protein is washed twice with phosphate buffer (10 ml of buffer per 106 ml of 0.36-0.55 saturated ammonium sulfate fraction) and the washes are combined with the original supernatant fluid. At this stage, the enzyme is stored in 0.70 saturated ammonium sulfate in batches of approximately 500,000 units and st,ored at -20”. From t,his point on the enzyme fractions are never allowed to freeze since 26-5Oyc of their activity is lost upon freezing and thawing. Chromatography on DEAE-Sephadex. Forty gm of DEAE-Sephadex A-50 is swelled in an excess of water. Fines are then removed by decantation 1520 t,imes. The Sephadex is then washed in a sintered-glass filter with 2 liters of 0.5 N HCl, followed quickly by several washes with water. The Sephadex is then washed with 2 liters of 0.5 N NaOH, again followed by large wash volumes of water. At this point,, fines are again removed by decantation. The material is then allowed to settle overnight to remove bubbles which formed during the washing periods. The washed mat,erial is adjusted to pH 5.7 by the addition of 1 N phosphoric acid. When the pH remains steady at 5.7, the DEAE-Sephadex is washed with 0.02 M phosphate buffer, pH 5.7, and then suspended in phosphate buffer of the same concentration. A 40 X 400 mm column is packed at room temperature to a height of 300 mm with the washed Sephadex under gravity flow. Only that mat,erial which settles on the column bed within 5 minutes is used for the packing. This is accomplished by pouring the washed Sephadex suspension back and forth between two beakers. In packing, the liquid head is allowed to stand only 5 minutes and
CRYSTALLINE
PYRUVATE
is then poured back int,o the beaker. B thin layer of glass wool is used to cover the top of the column. The DEAE-Sephadex column is charged with enzyme in the following manner. A bat.ch of 500,000 units of enzyme in 0.70 saturated ammonium sulfate is centrifuged for 30 minutes at 35,000g. The resulting precipitate is redissolved in 100-200 ml of 0.02 M potassium phosphate buffer, pH 5.7, and dialyzed 18 hours against a total volume of 6 liters of 0.02 M phosphate buffer, pH 5.7, in t.hree successive steps, using 2 liters of dialysis fluid for each step. The precipitate formed during dialysis is removed by centrifugation at 35,000g for 10 minutes and discarded. The supernatant fluid containing the flavoprotein is now ready for charging the column. The column is washed with 200 ml of 0.02 M potassium phosphate buffer, pH 5.7, and then cooled to 3-4”. The flow rate for the column should be at least ‘70-80 ml per hour at 3-4”. The dialyzed supernatant fluid containing the flavoprotein is applied to the DEAE-Sephadex column and the column is washed with a volume of 0.02 M phosphate buffer, pH 5.7, equal to one half the volume of the dialyzed supernatant fluid. The enzyme is eluted from the column with a linear gradient as described by Bock and Ling (10). The mixing flask contains 1100 ml of 0.02 M potassium phosphate buffer, pFI 5.7, and the reservoir contains 1100 ml of 0.3 M pot.assium phosphate buffer, pH 5.3. Fiveml fractions are collected. The enzyme usually is eluted in tubes 200-260. The enzyme is easily located by its yellow-green color and is usually the first of two closely associated colored peaks. The enzyme is assayed and the peak tubes are pooled so as to give an average specific activity of 1100 or more. The DEAE-Sephadex column fractionation should be completed in 18 hours or less since longer peritods of time lead to severe losses of enzyme activity. P~tami~ sztlfate fractionation. The pooled DEAE-Sephadex column fractions are brought to 0.70 ammonium sulfate saturation by t,he addition of 47.2 gm of ammonium sulfate per 100 ml of enzyme solution. The precipitate is removed by centrifugation at 35,000g for 20 minutes and redissolved in a volume of 0.02 M potassium phosphate buffer, pH $5.7, such as to give a final concentration of approximately 2O,OO(r30,000 units of enzyme per milliliter. The redissolved precipitate is then rapidly dialyzed against 50 volumes of 0.02 M potassium phosphate buffer, pH 5.7, in five successive steps using 10 volumes of dialysis fluid in each step. The precipitate formed during dialysis is removecl by centrifugation at 35,000g for 10 minutes and discarded.’ The supernatant fluid 7 If this precipitat,e is yellow, it is redissolved in 1 ml of 0.2 M potassium phosphate buffer pH,
171
OXIDASE
from the dialysis is treated with 1 volume of 2% protamine sulfate, pH 8, to precipitate the flavoprotein (if no precipitate forms immediately, the solution is dialyzed against distilled water to cause precipitation). The precipitate is removed by centrifugation at 35,OOOg for 10 minutes and suspended in approximately 1 ml of 0.2 M potassium phosphate buffer, pH 5.7, for each 10 ml of the above supernatant fluid. The insoluble precipitate is removed by centrifugation at 35,000g for 10 minutes and discarded. If the soluble flavoprotein remains in the supernatant fluid after protamine treatment and dialysis, it can be precipitated by a second protamine treatment. All the supernatant protamine eluate fractions are now pooled in preparation for the crystallization step. Cr@dZizatim. The combined supernatant fluid from the protamine fractionation step is slowly diluted with distilled water until a very slight. cloudiness appears. The solution is t,hen left to stand at -3” for 48 hours. At this point, impurities often precipitate with a small amount of the enzyme. The precipitate is removed by centrifugation at 35,000g for 10 minutes and, if necessary, the precipitate is eluted with 0.2 M potassium phosphate buffer, pH 5.7, to recover flavoprotein (which is added back to the mother liquor). The enzyme solution is then further diluted until a slight cloudiness appears (final buffer concentration at this point is 0.05-0.1 M). This solution is left to crystallize at -3” for a week or until the supernatant fluid is visibly clear of the yellow color. The enzyme preparation is then recrystallized in the same manner until constant specific activity crystals are obtained and the specific activity of the mother liquor equals that of the crystals. The results of a purification run are shown in Table I. RESULTS
Properties oj the crystalline protein. The enzyme crystallizes in large, regular, brittle, yellow rhombohedral crystals. In 2 or 3 days, crystals of 0.5 mm in length or larger were easily obtained from concentrated solutions of the pure flavoprotein. All physical studies were carried out on redissolved crystals that had been twicecrystallized. The enzyme moves as a single symmetrical peak in the ultracentrifuge at all concentrations from 2 to 11 mg per milli5.7, and the insoluble material centrifugation at 35,000g for supernatant solution is added supernatant fractions from the
is removed by 10 minutes. The to the combined protamine step.
172
WILLIAMS
AND
HAGER
TABLE PURIFICATION
I
OF FLAVIN
PYRUVATE
OXIDASE~ Enzyme activity
Fraction
Vohme (ml)
Crude extract Ammonium sulfate, 0.25-0.75, saturation Protamine sulfate supernatant fluid Ammonium sulfate, 0.36-0.55, saturation Supernatant fraction from heat treatment DEAE-Sephadex fractions Eluate fraction from protamine sulfate First crystallization Second crystallization Third crystallization Mother liquor of third crystallization
3800 2100
236,000 130,600
0.6 0.6
1698 1700
7.2 13.0
100 100
3480
33,200
1.2
1592
26.2
94
520
26,500
1.6
1200
48.0
70
598
10,000
1.5
1130
113.0
67
a 700 gm of E. coli
I
I
I
paste
I
I
Protein (mg)
2 ENZYME
~~~~~~~ (%)
582 294
1.5 1.3
754 587
1300.0 2000.0
45 35
5 5 4 12
134 69 50 19
1.3 1.2 1.2 1.2
469 418 300 118
3500.0 6000.0 6000.0 6000.0
28 25
was
used.
I
I
4 6 CONCENTRATION
~~~~~ units Specific activity (units/mg (1) 1 x 10-1) protein)
520 15
I
I
I
8 (mg/ml)
IO
8-
0
280/260 In@
FIG. 1. Dependence of sedimentation constant on concentration. Flavin pyruvate oxidase was dissolved in 0.2 M potassium phosphate buffer, pH 5.7, and the sedimentation constant &I”,~) was measured in the Beckman analytical ultracentrifuge, model E.
liter. The sedimentation constant ~$0,~ (extrapolated to infinite dilution and fully corrected) is 11.5 S (Fig. 1). The molecular weight determined by the Archibald modification of the sedimentation equilibrium method (11) is 265,000-285,000. The partial specific volume as determined with a lo-ml picnometer is 0.750 gm per milliliter. Spectral properties of the flavoprotein. Figure 2 shows the visible absorption spectrum of a solution of the crystalline enzyme. There are maxima at 370, 438, and 460 rnp, shoulders at 355 and 415 mM, and minima at, 390 and 455 rnw. The calculat.ed millimolar exbinction coefficients (per FAD prosthetic group) at 370, 438, and 460 mu are 11.3, 14.6, and 12.7 cnl-’ n1M-‘, respectively. The flavoprotein has a clearly defined absorption peak at 480 rnp, which is unusual since most flavoproteins have only a shoulder in this region. The reduced spectrum of the flavoprotein is also shown in Fig. 2. In the reduced form, all visible absorption bands except the 370 rnp peak disappear, and a considerable decrease in magnitude of this latter band is observed. The ultraviolet absorption
CRYSTALLINE
G “) 350
PYRUVATE
a ”
400
OXIDASE
a 0 0 ‘1’
450
WAVELENGTH
-
I
173
’
8 ” 500
m,u
The visible absorption spectra of the oxidized and reduced forms of pyruvate oxidase. The oxidized spectrum (Curve 1) was obtained by dissolving crystalline flavoprotein in standard phosphate buffer (1.67 mg protein/ml). The reduced spectrum (Curve 2) was obtained after the addition of 0.5 mg of sodiumhydrosulfite. FIG.
2.
spectrum shows a ratio of the absorption at 280 rnp to that at 260 rnp (280/260) of 1.2, which reflects the strong absorbancy of FAD at 260 mp. The millimolar extinction coefficient of the flavoprotein at 280 mp is 316. Flavin analysis. The flavin is removed from the (enzyme by heating the protein at 100” for 5 minutes. Since further treatment of the denatured protein with trypsin causes no further releaseof flavin, it is assumedthat the heat treatment releasesall of the flavin prosthetic group. The flavin released from the enzyme was identified as FAD by three different methods : (a) The spectrum of the released flavin corresponds to t’hat of a FAD standard and the ratio of the absorbancy at 260 rnp to t,hat at 450 mp (260/450) gave a value of 3.19 for th.e unknown as compared with 3.12 for the FAD standard. Beinert (12) reports the 260/450 ratios for FAD, FAIN, and riboflavin to be 3.27, 2.22, and 2.27, respect,ively. (b) Ascending chromat’ography in two
different solvent systems (t-butanol and water (60:40) and 5% Na,HP04 (13)) indicates that the unknown flavin from the enzyme and the FAD st,andard not only had the same R, value but moved together as one component. (c) The unknown flavin activates the apoflavin pyruvate oxidase from Lactobacillus delbrueckii. The L. delbrueckii enzyme utilizes FAD but does not respond to FMN or riboflavin (14). The FAD content, of the pyruvate oxidase was estimated by determining spectrophotometrically the amount of FAD liberated after the enzyme had been denatured by heating at 100” for 5 minutes. An extinction coefficient of 1.13 X lo4 Me’ cm-l at 448 rnp (12) was used for free FAD, and t’he results indicate that the oxidase contains 1 mole of FAD per 66,000 gm of enzyme. Since sedimentation equilibrium measurements indicate
a molecular
weight
265,000 for the flavoprotein,
value
of m
the flavin
174
WILLIAMS TABLE AMINO
ASP
LYS NH,
per mole of
enzyme
Average
22.hour hvdrolv-
45.hour hvdrolv-
6%hour hvdrolv-
180 240 200 265 169 214 129 43 60 17 75 117 78 52 71 117 350
176 237 198 261 161 212 127 31 53 12 69 119 76 48 70 116 370
176 228 193 256 164 209 127 14 37 9 70 110 77 27 70 112 424
-2&e-
Glu Gly Ala Val Leu Ile Serb Thrb 35 Cysb Met Pro Phe Tyr* IIis
II
ACID COMPOSITION OF FLAVIN PYRUVATE OXIDASE No. residues
Amino acida
AND
-ate-
l,;x,‘,‘“$;,
or
% ,,,, Weight
-zate177 235 197 261 165 212 128 56 70 20 71 115 77 60 70 115
7.63 11.37 4.24 6.99 6.16 9.04 5.46 1.84 2.67 0.78 3.51 4.21 4.27 3.69 3.62 5.55 -
a The amino acid analysis was carried out on approximately 5-mg aliquots of the lyophilized, dialyzed protein. A weighed sample was dissolved in 6 N hydrochloric acid, sealed in an evacuated tube, and held at 110” for the indicated time. Amino acid analysis was carried out on the Beckman model MS amino acid analyzer according to t,he method of Moore, Spackman, and Stein (15). h These amino acids were extrapolated to zero t,ime assuming first order decay.
analyses suggest there arc 4 moles of FAD per mole of enzyme. Amino acid analysis. An amino acid analysis was performed on the enzyme by t’he Moore, Spackman, and Stein method (15); a Beckman model MS amino acid analyzer was used (Table II). All amino acids are represented in essentially normal proportions. It will be noted, however, that the high ammonia content suggests that the dicarboxylic acids are present as amides, although this analysis is subject to some error since the nitrogen content by amino acid analysis (15.8 % nitrogen) is higher than that determined by microanalysis on dried enzyme (14.37 % -nitrogen). The partial specific volume, ‘I’ calculated from the individual amino acid content (0.744 gm per millilit.er) (II), is in good agreement with
HAGER
that calculated from density measurements (0.750 gm per milliliter). Reduction of the jlavoprotein. A direct measurement of the reduction of the flavoprotein by the substrate was made by spectral observation in a recording spectrophotometer. No change in the absorbancy at 33s mp could be observed unless pot’assium pyruvate, TPP, and Mg++ were all added in saturating amounts. At pH 6.0 and room temperature in the presence of 50 pmoles of potassium pyruvate, 10 I*moles of MgCL, and 0.1 @mole of TPP per milliliter, complete reduction of the flavoprotein occurred faster than the response time of the instrument (< 1 second). The enzyme substrate-co-factor complex is not autoxidizable; however, if substrate and co-factors are removed by dialysis, a rapid reoxidation of the enzyme by air is observed. The observed nonautoxidizability of the enzymesubstrate complex is not due to a constant reduction of the flavoprotein by excess substrate since there is no measurable oxygen uptake under the conditions where excess substrate is available. Enzymic activity. The flavoprotein pyruvate oxidase displays rather unique properties when various electron acceptors are used as oxidants for the reduced enzyme. As previously reported (2, 3), the activity of the flavoprotein can be increased 25-fold by various methods when dyes and artificial electron acceptors are used. Table III summarizes the ability of various activators to modify t’he activit’y of the flavoprotein touTard different electron acceptors. Surfaceactive agents increase the activity of the enzyme when ferricyanide and INT serve as electron acceptors. Among those surface-active agents tested were sodium lauryl sulfate, phosphat’idyl ethanolamine, ar-tocopherol phosphate, and succinate, whereas cr-tocopherol itself is not active. Non-ionic surface-active agents such as Tween 80 do not serve as activators. Partial digestion of the flavoprotein by proteolytic enzymes such as trypsin (a), chymotrypsin, or pepsin will produce similar results. An acetone extract of the particulate fraction (3) is also active; however, because of the extremely high activity of this extract and other evidence indicating a different mechanism of
CR.YSTALLINE
PYRUVATE
action (F. R. Williams and L. P. Hager, unpublished results), it is thought to be in a category separate from the surface-active agents. A lipo-protein fraction has been purified (Williams and Hager, unpublished results) from the solubilized particulate fraction (.5) and has been found to link t’he TrlBLE AWIVA’JYON INTEHACTION
OF
III
PYRUVATE OXIDASE FOR WITH VARIOUS ELECTRON ACCEPTOKS~ Electron acceptors
Activations
Fe+ cyanide
INT
TTC
Oxygen
1. Pyruvate oxidase alone 2. 1 plus sodiumlauryl sulfate 3. 1 preinoubated with t,rypsin 4. 1 plus acetone estractable factor 5. 1 plus lipoprotein G. 1 plus cytochromecontaining part’iculate fraction
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+ +
+ +
+ +
+
a Pyruvate oxidase was tested alone or in an activated .form for int)eraction wit,h various electron acceptors. For details concerning the trypsin activation reaction, see Ref. (2). For details concerning the preparation of the acetone extractable factor, see Ref. (3). For details concerning the preparation of the cytochrome-containing particulate fraction, see Refs. (4) and (5). The lipoprotein fraction is prepared from solubilized fractions derived from the particulate fraction by alcohol fractionation.
NUMBERS,
flavoprotein to TTC in addition to ferricyanide and INT, though not to molecular oxygen (Table III). There is evidence suggesting that the lipoprotein serves in an electron-transport capacity (Williams and Hager, unpublished results). Finally, the pyruvate oxidase alone is inactive with oxygen as the electron acceptor unless the particulate fraction or a solubilized fraction derived from this particulate fraction (4, 5) is added. Kinetic constants. In all cases an absolute requirement for TPP and potassium pyruvate was demonstrated for the reaction of the flavoprotein with the various electron acceptors. However, only a partial requirement for Mgt+ could be shown (see Table IV). The K, for pyruvate has previously been reported to be 1 X 1O-2 M. The K, for TPP of the pure crystalline enzyme is 1.2 X 1OF M, which is in good agreement with the value previously reported (1 X 1OF M) for less pure preparations (3). The K, for sodium lauryl sulfate for activation in the ferricyanide reaction is 3 X 10e5 M. Table IV lists some other properties of the activity of the enzyme. The maximum enzyme is 5-6 times greater in the ferricyanide-linked reaction than in the oxygen assay. The pH optimum for activity depends upon the assay and whether or not an activator is present. DISCUSSION
The results reported in this paper indicate that flavin pyruvate oxidase from E. coli is a Aavoprotein t,etranier consisting of
TABLE TURNOVER
175
OXIDASE
IV
pH OPTIMA, AND MG ++ DEPENDENCIES FOR PYRUVATE WITH DIFFERENT ELECTRON ACCEPTORS
OXIDASE
Electron acceptor
Ferricyanide”
Oxygen”
Property
Plus Na-lauryl sulfate, 1 x 10-a M 1. Turnover number 2. pH Optimum
53,000
0 See Materials
1,800 6.3
3. Mg++ Dependency, minus Mg++ ~x 100 plus Mg++ and
30
Methods
for
details
Minus activator
of the assay.
Plus particulate- fraction
Minus particulate fraction
9,000 5.2
12
0 5.8
39
-
176
WILLIAMS
66,000 molecular weight subunits. The flavoprotein binds two different prosthetic groups, thiamine pyrophosphate and flavinadenine dinucleotide. The crystalline flavin pyruvate oxidase exhibits several unusual properties which appear to be unique to this enzyme. The increased activity of the enzyme as reflected in the dramatic increase in turnover number when the oxidase is activated in a variety of ways merits further attention. Although activation of the flavoprotein can be accomplished by diverse methods, it is possible to suggest a common intcrpretat,ion with respect to mechanism of activation. All the reagents which lead to act,ivation of the flavoprotein fall into the category of causing protein denaturation. Thus it is possible to postulate that both trypsin hydrolysis and treatment of the flavoprotein with surface-active agents produces a limited denaturation of the enzyme which modifies the enzyme active site in a similar fashion. Thus a comparison of the native and activated forms of the flavoprotein could prove most fruitful in analyzing the act,ive site of this enzyme. The unusual properties of t,he flavoprotein also combined to make this enzyme a useful tool in studying the t#erminal electron transport system of the E. coli particulate fraction. The E. coli particulate fraction, as well as soluble enzyme preparations derived from the particulate fraction, catalyze the oxidat,ion of the flavoprotein using molecular
AND
HAGER
oxygen as t#he terminal electron acceptor. Apparent intermediates in the terminal electron sequence can be detected by using artificial electron acceptors. 1:EFERENCES 1. WILLIAMS, F. R., AND HAGER, L. I’., J. Biol. Chem. 236, PC 36 (1961). 2. HAGE~I, L. I’., J. Viol. Chem. 229, 251 (1957). 3. HAGER, L. P., J. Am. Chem. Sot. 79, 5575 (1957). 4. WILLIAMS, F. R., ASD HAGER, L. P., Biochim. Biophys. dcfa 38, 566 (1960). 5. DEEB,S.S., AND HAGER, L.P.,J.Biol. Chem. 239, 1021 (1964). 6. KORKES, S., Methods in Enzymol. I, 490 (1955). 7. GOUNARIS, A. I)., AND HAGER, L. P., J. Biol. Chem. 236, 1013 (1961). 8. STADT~IAN, E. R., NOVELLI, G. O., AND LIPMANX, F., J. Bid. Chem. 191, 365 (1951). 9. WARB~HG, O., AND CHRISTIAN, W., Biochem. 2.310, 384 (1911). 10. BOCK, R. M., ASD LING, NAN-SING, dnal. Chem. 26, 1513 (1954). 11. SCHACHMANN, H. K., Methods in Enzymol. IV, 33 (1957). 12. BEINERT, H., in “The Enzymes” (P. I>. Boyer, H. Lardy, and K. Myrbiich, eds.), Vol. 2A, p. 339. Academic Press, New York (1960). 13. KILGOUR, G. L., FELTON, S. P., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 79, 2254 (1957). 14. HAGER, L.P., GELLER,U.~CI.,AND LIPMANN, F., Federalion Proc. 13, 734 (1954). 15. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958).
OF BIOCHEMISTRY
Crystalline
AND
BIOPHYSICS
Flavin
Pyruvate
I. Isolation
B. Conant Laboratory, and the Biochemistry
168-176
WILLIAMS3
(1966)
Oxidase
and Properties
F. ROBERT James
116,
Escher-i&a
co/i
of the Flavoproteit? AND
LOWELL
Department of Chemistry, Harvard Division, Departwlent of Chemistry University of Illinois, Urbana, Received
from
May
P. HAGER4
University, Cambridge, Massachusetts and Chemical Engineering, Illinois
16, 1966
Pyruvate oxidase has been isolated from Escherichia coli in crystalline form and at, a high state of purity. Pyruvate oxidase binds both flavin-adenine dinucleotide and thiamine pyrophosphate as prosthetic groups. Molecular weight measurements indicate that the crystalline oxidase exists in solution as a tetramer. The minimal molecular weight of the oxidase, based on Aavin-adenine dinucleotide content, is 66,060, whereas the molecular weight of the oxidase, as measured by equilibrium sedimentation, is approximately 265,000. This paper reports the spectral properties, the amino acid content, the kinetic constants, and some of the enzymic activities of crystalline oxidase.
Previous communications (l-5) from this laboratory have reported the presence of a flavin pyruvate oxidase system in Escherichia coli. This enzyme system catalyzes the oxidative decarboxylation of pyruvate to acetate and carbon dioxide according to Eq. (1): CH,COCOOH
+ 36 02
TPP,Mg++, terminal
As indicated in Eq. (I), TPP,5 Ng++, and a cytochrome-containing terminal electron transport system are required (1, 4, .5) in addition to the flavoprotein for catalysis in this reaction. The terminal electron system of E. coli can be obtained in the form of a particulate fraction which sediments at high gravitational forces (4). This flavoproteincytochrome system for pyruvate oxidation is distinct and separate from t’he CoA, DPNf, and lipoic acid-pyruvate dehydrogenase complex which is also present in E. coli (6, 7). Ferricyanide can replace oxygen as an electron acceptor in Eq. (1). In this case, the flavoprotein reacts sluggishly with ferricyanide in t’he absence of the cytochromecontaining particulate fraction. However, the rate of the ferricyanide-linked reaction can be increased approximately 25-fold by several means. Perincubation of the flavoprotein with trypsin (2), or the addition of certain surface-active agents, such as
electron
flavoprotein transport
plus
a
system CH&OOH
> + CO2
1 Dedicated to Luis F. Leloir on the occasion of his sixtieth birthday. 2 This investigation was supported by a grant (RG 7768) from the National Institutes of Health. Portions of this work were taken from a thesis submitted by F. Robert Williams in partial satisfaction of requirements for a degree of Doctor of Philosophy, Harvard University. A preliminary report of this work has appeared (1). 3 This author held a predoctoral fellowship from the National Institutes of Health at the time of investigation. Present address: Service de Biochimie, Centre d’etudes Nucleaires de SaclayGif-sur-Yvette, S. et 0. France. 4 Reprint requests should be addressed to this author at Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois.
6 Abbreviations used: DPN+, diphosphopyridine nucleotide; FAD, flavin-adenine dinucleotide; FMN, flavin mononucleotide; INT, 2-(piodophenyl)-3-p-nitrophenyld-phenyl tetrazolium chloride; TPP, thiamine pyrophosphate; TTC, triphenyl tetrazolium chloride. 168
CRYSTALLINE
PYRUVATE
sodium lauryl sulfate, or the addition of the particulate fraction (4) or solubilized preparations derived from t’he particulate fraction (5) markedly enhance the activity of the flavoprotein with ferricyanide. Acetone extracts of the particulate fraction also activate the flavoprotein in the ferricyanidelinked react,ion (3). We shall present in this paper the final purificat,ion and cryst’allization of the flavoprotein component of this system and report on some of the physical and catalytic propert’ies of the enzyme. M.$TERIALS
AND
METHODS
Enzyme assay. The flavoprotein is routinely assayed by measuring carbon dioxide release from pyruvate with the ferricyanide serving as the electron acceptor. The assay reaction mixture contains 1ClOrmoles of potassium phosphate buffer, pH 6.0; 0.1 Imole of TPP; 10 @moles of MgCl:; 50 pmoles of potassium pyruvate; 0.5 mg of ovalbumin; 1 pmole of sodium lauryl sulfate; 25 pmoles of potassium ferricyanide; and sufficient enzyme to catalyze the evolution of 0.3-3 pmoles of carbon dioxide per 5.minute interval. The reaction is started by t,he addition of ferricyanide from the sidearm of the Warburg cup. The incubation temperature is 31”. One unit of enzymic activity in the ferricyanide assay is defined as the evolution of 1 rmole of carbon dioxide per 30 minutes under the assay conditions described above. The rate of carbon dioxide evolution is estimated from the first 10 minutes of t.he reaction. Specific activity is defined as units per milligram of protein. Oxygen assay. The flavin pyruvate oxidase is nonautoxidizable in the form of its enzymesubstrate complex; however, oxygen uptake can be observed in a coupled system when the oxidase is supplemented with a particulate fraction derived from the cell envelope. This assay, which has been previously described (2), can be used both to estimate the act,ivity of flavoproteiu in the presence of an excess of the particulate fraction and the enzymic activity of the particulate fraction in the ‘presence of an excess of flavoprotein (4). Protein determinations. Protein concentrations were estimated by three different methods depending on the purity of the enzyme. If the ratio of 280 to 260 mp absorption was 0.6 or less, the protein concentrat:lon was measured by the trichloroacetic acid precipitation method (8). The protein concentrat[ons of fractions having a 280/260 ratio of 0.6 or more were measured by the WarburgChristian method (9). The concentration of crystalline enzyme was obtained from the absorption
169
OXIDASE
at 280 rn/* with a multiplication factor of 0.84 for the conversion of absorbance (1 cm lightpath) to milligram of protein per milliliter. The conversion factor was based on dry-weight det,erminations. ~1Z&riaZs. Commercial preparations of FAD, FMN, riboflavin, and TPP were obtained from the Sigma Chemical Corp. Potassium pyruvate, ovalbumin, and protamine sulfate were products of Nutritional Biochemicals Corp. DEAE-Sephades was obtained from Pharmacia Co. Flavin-adenine dinucleotide and flavin mononucleotide standards were prepared by ascending paper chromatography of commercial FAD and FMN; a solvent system composed of t-butanol and water (60:40) was used. The spots corresponding t,o FAD and FMN were cut out, eluted in the cold, checked by spectral analysis, and stored at 4” in the dark. All operations involving free flavins were carried out wherever possible at 4” in the dark.
Enzyme PuriJication Growth medizcm. The cells are grown on a medium containing 0.2% NH&l; 0.4% glucose; 0.25% sodium glutamate; 0.0005~o yeast extract; 0.150/, KH?PO*; 1.35% NazHPOa; 0.02% MgSOa.7 H,O; O.OOl’% CaC12; and 0.00005% FeSOI.7 H?O. The phosphate, glutamate, and glucose are sterilized in separate containers and added to the sterile salt solution. When the acetate mutant is used as the source of enzyme, the medium is further supplemented with 20 pmoles of potassium acetate per milliliter. The cells are grown under forced aeration, either in lo-liter batches in a New Brunswick fermentor, or in loo-liter batches in a 5C-gallon fermentor manufactured by Stainless and Steel Products Co. Inoculum. Escherichia coli, strain B, Crookes, W, or the acetate-requiring mutant 191-6 (7) derived from strain W, is grown on a complex medium containing 1% tryptone, 1% yeast extract, 0.59/ KZHPOI, and 0.3% glucose. Actively growing cells (log phase) are inoculated at a 0.5% level into the growth medium. Growth. Cells are grown to the stationary phase (15-20 hours) and are then harvested on a Sharples centrifuge. The yield is approximately 10 gm of cell paste per liter of culture medium. Preparation of cell extract. Six hundred gm of cell paste is suspended in 500 ml of 0.02 M potassium phosphate buffer, pH 7,6 and mixed thoroughly in a Servall omnimixer. Six hundred ml of washed glass beads (Minneapolis Mining and 0 Unless otherwise to buffer, phosphate buffer refer to 0.02 pH 7.
noted, subsequent references buffer, or standard phosphate M potassium phosphate buffer,
170
WILLIAMS
Manufacturing Co., Minnesota), 120 p in diameter, are added to the cell suspension. The cell-glass bead mixture is then ground in an Eppenbach colloid mill (rotor-stator setting of 0.030) for 30 minutes at 15-20” with continuous recycling. The resulting slurry is centrifuged at 15,OOOg for 10 minutes to remove the glass beads. The supernatant fluid (supernatant 1) is saved. The precipitate (together with the glass beads) is washed by resuspension in 1000 ml of phosphate buffer, and is then centrifuged at 15,000g for 10 minutes. The supernatant fluid (supernat,ant 2) is again saved. The precipitate (consisting mostly of glass beads) is again resuspended in 1000 ml of phosphate buffer and allowed to sediment for 1 hour. The supernatant fluid (supernatant 3) is decanted and saved. Supernatants 1 and 2 are combined and centrifuged at 15,000g for 1 hour. Three layers are formed during the centrifugation: a clear liquid phase; a dense, viscous particulate phase; and a precipitate of cell debris. The liquid phase and the particulate phase are removed toget,her and saved (supernatant 4). The precipitate of cell debris is washed by resuspension in supernatant 3 and is centrifuged at 15,000g for 1 hour. The precipitate is discarded. The supernatant fluid (supernatant 5) is removed and combined with supernatant 4 t’o yield a crude extract. First ammonium suljate precipitation. The crude extract is brought to 0.25 ammonium sulfate saturation by the addition of 14.4 gm of ammonium sulfate per 100 ml of crude extract. The suspension is stirred for 4 hours. The precipitate is removed by centrifugation at 15,OOOg for 75 minutes. This precipitate is saved and is used for the preparation of crystalline cytochrome bl (5). The supernatant fluid is brought to 0.75 ammonium sulfate saturation by the addition of 34.4 gm of ammonium sulfate per 100 ml of supernatant fluid. The precipitate is removed by centrifugation at 15,000g for 30 minutes and redissolved in a volume of buffer equal to one-half the volume of the initial crude extract. Removal of nucleic acid. The 0.25-0.75 saturated ammonium sulfate fraction is adjusted to pH 6 by the addition of 1 N acetic acid. Nucleic acid and inactive protein are precipitated by the slow addition of an equal volume of 2$!$ protamine sulfate, pH 6. The resulting mixture is dialyzed overnight against a total of 20 volumes of phosphate buffer. The insoluble protamine nucleate, which precipitates during dialysis, is removed by centrifugation at 15,000g for 20 minutes and discarded. At this stage the ratio of ultraviolet absorption at 280 rnp and 260 mp (280/260) of the supernatant solution should be above 1, although successful purifications have been carried out with fractions having a ratio of 0.8.
AND
HAGER
Second ammonium suljaie precipitation. The supernatant fluid from the protamine sulfate treatment is brought to 0.36 ammonium sulfate saturation by the addition of 25.2 gm of ammonium sulfate per 100 ml of supernatant fluid. After stirring for 2-3 hours, the resulting precipitate is removed by centrifugation at 15,000g for 30 minutes and discarded. The supernntant fluid obtained from the 0.36 saturated ammonium sulfate fraction is brought to 0.55 ammonium sulfate saturation by the addition of 12.1 gm of ammonium sulfate per 100 ml of supernntant fluid and stirred for 2-3 hours. The precipit,ate is removed by centrifugation at 15,OoOg for 30 minutes and redissolved in a volume of buffer equal to 0.06 volume of the crude extract. Heat precipitation. The 0.36-0.55 saturated ammonium sulfate fraction is rapidly warmed to 60’ in a 70-80” bath with vigorous stirring and then held at 60” in a 60” bath for 5 minutes. The heattreated fraction is rapidly cooled in an ice bath and the precipitate of denatured protein is removed by cent.rifugation at 35,000g for 1 hour. The precipitate of denatured protein is washed twice with phosphate buffer (10 ml of buffer per 106 ml of 0.36-0.55 saturated ammonium sulfate fraction) and the washes are combined with the original supernatant fluid. At this stage, the enzyme is stored in 0.70 saturated ammonium sulfate in batches of approximately 500,000 units and st,ored at -20”. From t,his point on the enzyme fractions are never allowed to freeze since 26-5Oyc of their activity is lost upon freezing and thawing. Chromatography on DEAE-Sephadex. Forty gm of DEAE-Sephadex A-50 is swelled in an excess of water. Fines are then removed by decantation 1520 t,imes. The Sephadex is then washed in a sintered-glass filter with 2 liters of 0.5 N HCl, followed quickly by several washes with water. The Sephadex is then washed with 2 liters of 0.5 N NaOH, again followed by large wash volumes of water. At this point,, fines are again removed by decantation. The material is then allowed to settle overnight to remove bubbles which formed during the washing periods. The washed mat,erial is adjusted to pH 5.7 by the addition of 1 N phosphoric acid. When the pH remains steady at 5.7, the DEAE-Sephadex is washed with 0.02 M phosphate buffer, pH 5.7, and then suspended in phosphate buffer of the same concentration. A 40 X 400 mm column is packed at room temperature to a height of 300 mm with the washed Sephadex under gravity flow. Only that mat,erial which settles on the column bed within 5 minutes is used for the packing. This is accomplished by pouring the washed Sephadex suspension back and forth between two beakers. In packing, the liquid head is allowed to stand only 5 minutes and
CRYSTALLINE
PYRUVATE
is then poured back int,o the beaker. B thin layer of glass wool is used to cover the top of the column. The DEAE-Sephadex column is charged with enzyme in the following manner. A bat.ch of 500,000 units of enzyme in 0.70 saturated ammonium sulfate is centrifuged for 30 minutes at 35,000g. The resulting precipitate is redissolved in 100-200 ml of 0.02 M potassium phosphate buffer, pH 5.7, and dialyzed 18 hours against a total volume of 6 liters of 0.02 M phosphate buffer, pH 5.7, in t.hree successive steps, using 2 liters of dialysis fluid for each step. The precipitate formed during dialysis is removed by centrifugation at 35,000g for 10 minutes and discarded. The supernatant fluid containing the flavoprotein is now ready for charging the column. The column is washed with 200 ml of 0.02 M potassium phosphate buffer, pH 5.7, and then cooled to 3-4”. The flow rate for the column should be at least ‘70-80 ml per hour at 3-4”. The dialyzed supernatant fluid containing the flavoprotein is applied to the DEAE-Sephadex column and the column is washed with a volume of 0.02 M phosphate buffer, pH 5.7, equal to one half the volume of the dialyzed supernatant fluid. The enzyme is eluted from the column with a linear gradient as described by Bock and Ling (10). The mixing flask contains 1100 ml of 0.02 M potassium phosphate buffer, pFI 5.7, and the reservoir contains 1100 ml of 0.3 M pot.assium phosphate buffer, pH 5.3. Fiveml fractions are collected. The enzyme usually is eluted in tubes 200-260. The enzyme is easily located by its yellow-green color and is usually the first of two closely associated colored peaks. The enzyme is assayed and the peak tubes are pooled so as to give an average specific activity of 1100 or more. The DEAE-Sephadex column fractionation should be completed in 18 hours or less since longer peritods of time lead to severe losses of enzyme activity. P~tami~ sztlfate fractionation. The pooled DEAE-Sephadex column fractions are brought to 0.70 ammonium sulfate saturation by t,he addition of 47.2 gm of ammonium sulfate per 100 ml of enzyme solution. The precipitate is removed by centrifugation at 35,000g for 20 minutes and redissolved in a volume of 0.02 M potassium phosphate buffer, pH $5.7, such as to give a final concentration of approximately 2O,OO(r30,000 units of enzyme per milliliter. The redissolved precipitate is then rapidly dialyzed against 50 volumes of 0.02 M potassium phosphate buffer, pH 5.7, in five successive steps using 10 volumes of dialysis fluid in each step. The precipitate formed during dialysis is removecl by centrifugation at 35,000g for 10 minutes and discarded.’ The supernatant fluid 7 If this precipitat,e is yellow, it is redissolved in 1 ml of 0.2 M potassium phosphate buffer pH,
171
OXIDASE
from the dialysis is treated with 1 volume of 2% protamine sulfate, pH 8, to precipitate the flavoprotein (if no precipitate forms immediately, the solution is dialyzed against distilled water to cause precipitation). The precipitate is removed by centrifugation at 35,OOOg for 10 minutes and suspended in approximately 1 ml of 0.2 M potassium phosphate buffer, pH 5.7, for each 10 ml of the above supernatant fluid. The insoluble precipitate is removed by centrifugation at 35,000g for 10 minutes and discarded. If the soluble flavoprotein remains in the supernatant fluid after protamine treatment and dialysis, it can be precipitated by a second protamine treatment. All the supernatant protamine eluate fractions are now pooled in preparation for the crystallization step. Cr@dZizatim. The combined supernatant fluid from the protamine fractionation step is slowly diluted with distilled water until a very slight. cloudiness appears. The solution is t,hen left to stand at -3” for 48 hours. At this point, impurities often precipitate with a small amount of the enzyme. The precipitate is removed by centrifugation at 35,000g for 10 minutes and, if necessary, the precipitate is eluted with 0.2 M potassium phosphate buffer, pH 5.7, to recover flavoprotein (which is added back to the mother liquor). The enzyme solution is then further diluted until a slight cloudiness appears (final buffer concentration at this point is 0.05-0.1 M). This solution is left to crystallize at -3” for a week or until the supernatant fluid is visibly clear of the yellow color. The enzyme preparation is then recrystallized in the same manner until constant specific activity crystals are obtained and the specific activity of the mother liquor equals that of the crystals. The results of a purification run are shown in Table I. RESULTS
Properties oj the crystalline protein. The enzyme crystallizes in large, regular, brittle, yellow rhombohedral crystals. In 2 or 3 days, crystals of 0.5 mm in length or larger were easily obtained from concentrated solutions of the pure flavoprotein. All physical studies were carried out on redissolved crystals that had been twicecrystallized. The enzyme moves as a single symmetrical peak in the ultracentrifuge at all concentrations from 2 to 11 mg per milli5.7, and the insoluble material centrifugation at 35,000g for supernatant solution is added supernatant fractions from the
is removed by 10 minutes. The to the combined protamine step.
172
WILLIAMS
AND
HAGER
TABLE PURIFICATION
I
OF FLAVIN
PYRUVATE
OXIDASE~ Enzyme activity
Fraction
Vohme (ml)
Crude extract Ammonium sulfate, 0.25-0.75, saturation Protamine sulfate supernatant fluid Ammonium sulfate, 0.36-0.55, saturation Supernatant fraction from heat treatment DEAE-Sephadex fractions Eluate fraction from protamine sulfate First crystallization Second crystallization Third crystallization Mother liquor of third crystallization
3800 2100
236,000 130,600
0.6 0.6
1698 1700
7.2 13.0
100 100
3480
33,200
1.2
1592
26.2
94
520
26,500
1.6
1200
48.0
70
598
10,000
1.5
1130
113.0
67
a 700 gm of E. coli
I
I
I
paste
I
I
Protein (mg)
2 ENZYME
~~~~~~~ (%)
582 294
1.5 1.3
754 587
1300.0 2000.0
45 35
5 5 4 12
134 69 50 19
1.3 1.2 1.2 1.2
469 418 300 118
3500.0 6000.0 6000.0 6000.0
28 25
was
used.
I
I
4 6 CONCENTRATION
~~~~~ units Specific activity (units/mg (1) 1 x 10-1) protein)
520 15
I
I
I
8 (mg/ml)
IO
8-
0
280/260 In@
FIG. 1. Dependence of sedimentation constant on concentration. Flavin pyruvate oxidase was dissolved in 0.2 M potassium phosphate buffer, pH 5.7, and the sedimentation constant &I”,~) was measured in the Beckman analytical ultracentrifuge, model E.
liter. The sedimentation constant ~$0,~ (extrapolated to infinite dilution and fully corrected) is 11.5 S (Fig. 1). The molecular weight determined by the Archibald modification of the sedimentation equilibrium method (11) is 265,000-285,000. The partial specific volume as determined with a lo-ml picnometer is 0.750 gm per milliliter. Spectral properties of the flavoprotein. Figure 2 shows the visible absorption spectrum of a solution of the crystalline enzyme. There are maxima at 370, 438, and 460 rnp, shoulders at 355 and 415 mM, and minima at, 390 and 455 rnw. The calculat.ed millimolar exbinction coefficients (per FAD prosthetic group) at 370, 438, and 460 mu are 11.3, 14.6, and 12.7 cnl-’ n1M-‘, respectively. The flavoprotein has a clearly defined absorption peak at 480 rnp, which is unusual since most flavoproteins have only a shoulder in this region. The reduced spectrum of the flavoprotein is also shown in Fig. 2. In the reduced form, all visible absorption bands except the 370 rnp peak disappear, and a considerable decrease in magnitude of this latter band is observed. The ultraviolet absorption
CRYSTALLINE
G “) 350
PYRUVATE
a ”
400
OXIDASE
a 0 0 ‘1’
450
WAVELENGTH
-
I
173
’
8 ” 500
m,u
The visible absorption spectra of the oxidized and reduced forms of pyruvate oxidase. The oxidized spectrum (Curve 1) was obtained by dissolving crystalline flavoprotein in standard phosphate buffer (1.67 mg protein/ml). The reduced spectrum (Curve 2) was obtained after the addition of 0.5 mg of sodiumhydrosulfite. FIG.
2.
spectrum shows a ratio of the absorption at 280 rnp to that at 260 rnp (280/260) of 1.2, which reflects the strong absorbancy of FAD at 260 mp. The millimolar extinction coefficient of the flavoprotein at 280 mp is 316. Flavin analysis. The flavin is removed from the (enzyme by heating the protein at 100” for 5 minutes. Since further treatment of the denatured protein with trypsin causes no further releaseof flavin, it is assumedthat the heat treatment releasesall of the flavin prosthetic group. The flavin released from the enzyme was identified as FAD by three different methods : (a) The spectrum of the released flavin corresponds to t’hat of a FAD standard and the ratio of the absorbancy at 260 rnp to t,hat at 450 mp (260/450) gave a value of 3.19 for th.e unknown as compared with 3.12 for the FAD standard. Beinert (12) reports the 260/450 ratios for FAD, FAIN, and riboflavin to be 3.27, 2.22, and 2.27, respect,ively. (b) Ascending chromat’ography in two
different solvent systems (t-butanol and water (60:40) and 5% Na,HP04 (13)) indicates that the unknown flavin from the enzyme and the FAD st,andard not only had the same R, value but moved together as one component. (c) The unknown flavin activates the apoflavin pyruvate oxidase from Lactobacillus delbrueckii. The L. delbrueckii enzyme utilizes FAD but does not respond to FMN or riboflavin (14). The FAD content, of the pyruvate oxidase was estimated by determining spectrophotometrically the amount of FAD liberated after the enzyme had been denatured by heating at 100” for 5 minutes. An extinction coefficient of 1.13 X lo4 Me’ cm-l at 448 rnp (12) was used for free FAD, and t’he results indicate that the oxidase contains 1 mole of FAD per 66,000 gm of enzyme. Since sedimentation equilibrium measurements indicate
a molecular
weight
265,000 for the flavoprotein,
value
of m
the flavin
174
WILLIAMS TABLE AMINO
ASP
LYS NH,
per mole of
enzyme
Average
22.hour hvdrolv-
45.hour hvdrolv-
6%hour hvdrolv-
180 240 200 265 169 214 129 43 60 17 75 117 78 52 71 117 350
176 237 198 261 161 212 127 31 53 12 69 119 76 48 70 116 370
176 228 193 256 164 209 127 14 37 9 70 110 77 27 70 112 424
-2&e-
Glu Gly Ala Val Leu Ile Serb Thrb 35 Cysb Met Pro Phe Tyr* IIis
II
ACID COMPOSITION OF FLAVIN PYRUVATE OXIDASE No. residues
Amino acida
AND
-ate-
l,;x,‘,‘“$;,
or
% ,,,, Weight
-zate177 235 197 261 165 212 128 56 70 20 71 115 77 60 70 115
7.63 11.37 4.24 6.99 6.16 9.04 5.46 1.84 2.67 0.78 3.51 4.21 4.27 3.69 3.62 5.55 -
a The amino acid analysis was carried out on approximately 5-mg aliquots of the lyophilized, dialyzed protein. A weighed sample was dissolved in 6 N hydrochloric acid, sealed in an evacuated tube, and held at 110” for the indicated time. Amino acid analysis was carried out on the Beckman model MS amino acid analyzer according to t,he method of Moore, Spackman, and Stein (15). h These amino acids were extrapolated to zero t,ime assuming first order decay.
analyses suggest there arc 4 moles of FAD per mole of enzyme. Amino acid analysis. An amino acid analysis was performed on the enzyme by t’he Moore, Spackman, and Stein method (15); a Beckman model MS amino acid analyzer was used (Table II). All amino acids are represented in essentially normal proportions. It will be noted, however, that the high ammonia content suggests that the dicarboxylic acids are present as amides, although this analysis is subject to some error since the nitrogen content by amino acid analysis (15.8 % nitrogen) is higher than that determined by microanalysis on dried enzyme (14.37 % -nitrogen). The partial specific volume, ‘I’ calculated from the individual amino acid content (0.744 gm per millilit.er) (II), is in good agreement with
HAGER
that calculated from density measurements (0.750 gm per milliliter). Reduction of the jlavoprotein. A direct measurement of the reduction of the flavoprotein by the substrate was made by spectral observation in a recording spectrophotometer. No change in the absorbancy at 33s mp could be observed unless pot’assium pyruvate, TPP, and Mg++ were all added in saturating amounts. At pH 6.0 and room temperature in the presence of 50 pmoles of potassium pyruvate, 10 I*moles of MgCL, and 0.1 @mole of TPP per milliliter, complete reduction of the flavoprotein occurred faster than the response time of the instrument (< 1 second). The enzyme substrate-co-factor complex is not autoxidizable; however, if substrate and co-factors are removed by dialysis, a rapid reoxidation of the enzyme by air is observed. The observed nonautoxidizability of the enzymesubstrate complex is not due to a constant reduction of the flavoprotein by excess substrate since there is no measurable oxygen uptake under the conditions where excess substrate is available. Enzymic activity. The flavoprotein pyruvate oxidase displays rather unique properties when various electron acceptors are used as oxidants for the reduced enzyme. As previously reported (2, 3), the activity of the flavoprotein can be increased 25-fold by various methods when dyes and artificial electron acceptors are used. Table III summarizes the ability of various activators to modify t’he activit’y of the flavoprotein touTard different electron acceptors. Surfaceactive agents increase the activity of the enzyme when ferricyanide and INT serve as electron acceptors. Among those surface-active agents tested were sodium lauryl sulfate, phosphat’idyl ethanolamine, ar-tocopherol phosphate, and succinate, whereas cr-tocopherol itself is not active. Non-ionic surface-active agents such as Tween 80 do not serve as activators. Partial digestion of the flavoprotein by proteolytic enzymes such as trypsin (a), chymotrypsin, or pepsin will produce similar results. An acetone extract of the particulate fraction (3) is also active; however, because of the extremely high activity of this extract and other evidence indicating a different mechanism of
CR.YSTALLINE
PYRUVATE
action (F. R. Williams and L. P. Hager, unpublished results), it is thought to be in a category separate from the surface-active agents. A lipo-protein fraction has been purified (Williams and Hager, unpublished results) from the solubilized particulate fraction (.5) and has been found to link t’he TrlBLE AWIVA’JYON INTEHACTION
OF
III
PYRUVATE OXIDASE FOR WITH VARIOUS ELECTRON ACCEPTOKS~ Electron acceptors
Activations
Fe+ cyanide
INT
TTC
Oxygen
1. Pyruvate oxidase alone 2. 1 plus sodiumlauryl sulfate 3. 1 preinoubated with t,rypsin 4. 1 plus acetone estractable factor 5. 1 plus lipoprotein G. 1 plus cytochromecontaining part’iculate fraction
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+ +
+ +
+ +
+
a Pyruvate oxidase was tested alone or in an activated .form for int)eraction wit,h various electron acceptors. For details concerning the trypsin activation reaction, see Ref. (2). For details concerning the preparation of the acetone extractable factor, see Ref. (3). For details concerning the preparation of the cytochrome-containing particulate fraction, see Refs. (4) and (5). The lipoprotein fraction is prepared from solubilized fractions derived from the particulate fraction by alcohol fractionation.
NUMBERS,
flavoprotein to TTC in addition to ferricyanide and INT, though not to molecular oxygen (Table III). There is evidence suggesting that the lipoprotein serves in an electron-transport capacity (Williams and Hager, unpublished results). Finally, the pyruvate oxidase alone is inactive with oxygen as the electron acceptor unless the particulate fraction or a solubilized fraction derived from this particulate fraction (4, 5) is added. Kinetic constants. In all cases an absolute requirement for TPP and potassium pyruvate was demonstrated for the reaction of the flavoprotein with the various electron acceptors. However, only a partial requirement for Mgt+ could be shown (see Table IV). The K, for pyruvate has previously been reported to be 1 X 1O-2 M. The K, for TPP of the pure crystalline enzyme is 1.2 X 1OF M, which is in good agreement with the value previously reported (1 X 1OF M) for less pure preparations (3). The K, for sodium lauryl sulfate for activation in the ferricyanide reaction is 3 X 10e5 M. Table IV lists some other properties of the activity of the enzyme. The maximum enzyme is 5-6 times greater in the ferricyanide-linked reaction than in the oxygen assay. The pH optimum for activity depends upon the assay and whether or not an activator is present. DISCUSSION
The results reported in this paper indicate that flavin pyruvate oxidase from E. coli is a Aavoprotein t,etranier consisting of
TABLE TURNOVER
175
OXIDASE
IV
pH OPTIMA, AND MG ++ DEPENDENCIES FOR PYRUVATE WITH DIFFERENT ELECTRON ACCEPTORS
OXIDASE
Electron acceptor
Ferricyanide”
Oxygen”
Property
Plus Na-lauryl sulfate, 1 x 10-a M 1. Turnover number 2. pH Optimum
53,000
0 See Materials
1,800 6.3
3. Mg++ Dependency, minus Mg++ ~x 100 plus Mg++ and
30
Methods
for
details
Minus activator
of the assay.
Plus particulate- fraction
Minus particulate fraction
9,000 5.2
12
0 5.8
39
-
176
WILLIAMS
66,000 molecular weight subunits. The flavoprotein binds two different prosthetic groups, thiamine pyrophosphate and flavinadenine dinucleotide. The crystalline flavin pyruvate oxidase exhibits several unusual properties which appear to be unique to this enzyme. The increased activity of the enzyme as reflected in the dramatic increase in turnover number when the oxidase is activated in a variety of ways merits further attention. Although activation of the flavoprotein can be accomplished by diverse methods, it is possible to suggest a common intcrpretat,ion with respect to mechanism of activation. All the reagents which lead to act,ivation of the flavoprotein fall into the category of causing protein denaturation. Thus it is possible to postulate that both trypsin hydrolysis and treatment of the flavoprotein with surface-active agents produces a limited denaturation of the enzyme which modifies the enzyme active site in a similar fashion. Thus a comparison of the native and activated forms of the flavoprotein could prove most fruitful in analyzing the act,ive site of this enzyme. The unusual properties of t,he flavoprotein also combined to make this enzyme a useful tool in studying the t#erminal electron transport system of the E. coli particulate fraction. The E. coli particulate fraction, as well as soluble enzyme preparations derived from the particulate fraction, catalyze the oxidat,ion of the flavoprotein using molecular
AND
HAGER
oxygen as t#he terminal electron acceptor. Apparent intermediates in the terminal electron sequence can be detected by using artificial electron acceptors. 1:EFERENCES 1. WILLIAMS, F. R., AND HAGER, L. I’., J. Biol. Chem. 236, PC 36 (1961). 2. HAGE~I, L. I’., J. Viol. Chem. 229, 251 (1957). 3. HAGER, L. P., J. Am. Chem. Sot. 79, 5575 (1957). 4. WILLIAMS, F. R., ASD HAGER, L. P., Biochim. Biophys. dcfa 38, 566 (1960). 5. DEEB,S.S., AND HAGER, L.P.,J.Biol. Chem. 239, 1021 (1964). 6. KORKES, S., Methods in Enzymol. I, 490 (1955). 7. GOUNARIS, A. I)., AND HAGER, L. P., J. Biol. Chem. 236, 1013 (1961). 8. STADT~IAN, E. R., NOVELLI, G. O., AND LIPMANX, F., J. Bid. Chem. 191, 365 (1951). 9. WARB~HG, O., AND CHRISTIAN, W., Biochem. 2.310, 384 (1911). 10. BOCK, R. M., ASD LING, NAN-SING, dnal. Chem. 26, 1513 (1954). 11. SCHACHMANN, H. K., Methods in Enzymol. IV, 33 (1957). 12. BEINERT, H., in “The Enzymes” (P. I>. Boyer, H. Lardy, and K. Myrbiich, eds.), Vol. 2A, p. 339. Academic Press, New York (1960). 13. KILGOUR, G. L., FELTON, S. P., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 79, 2254 (1957). 14. HAGER, L.P., GELLER,U.~CI.,AND LIPMANN, F., Federalion Proc. 13, 734 (1954). 15. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958).