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Yeast inorganic pyrophosphatase
ARCHIVb:S
OF
BIOCHEMISTRY
.\ND
Yeast IV. Purification,
Quaternary
inorganic
of Biochemisky,
714-725
(1972)
Pyrophosphatase
Structure,
J. W. RIDLINGTON, Department
163,
HIOPHYSICS
and Evidence
Y. YANG, Purdue Received
AND
Ilniversily, July
for Strongly
Bound Mg’+’
L. G. BUTLER Lafayette,
Indiana
g7907
5, 1972
Yeast inorganic pyrophosphatase has been purified to apparent homogeneity by salt fractionation and chromatographic techniques avoiding the use of organic solvents. The enzyme is obtained in stable crystalline form. The molecular weight of the native eueyme is 71,000 & 2000, and it consists of two subunits of similar, if not identical, mass and charge. The enzyme as isolated contains approximately 1 mole of strongly bound Mg2+ per mole of active dimer; certain chelating agents cause an enhancement of the intrinsic protein fluorescence, presumably by complexing with this Mgz+ while it, remains bound to the enzyme. The Mg2+ can be partially removed by chelators under specified conditious; the metal-free enzyme appears to be inactive, and is much more susceptible to proteolytic digestion and dissociation to monomers than is the native enzyme. The activity of met,al-free enzyme may be rapidly restored suggest that Zn2+ or other ions may replace the in the presence of Mg 2+; the results strongly bound M$+, with consequent loss of activity.
The inorganic pyrophosphatasc (pyrophosphatase phosphohydrolasc EC 3.6.1.1) reaction is perhaps the simplest system, in terms of the chemical structures involved, for enzymatic utilization of physiological polyphosphates, and is, therefore, an attractive model for mccha,nistic studios. For many years the only available pure (crystalline) pyrophosphatase was that obtained from baker’s yeast using the procedura describrd by Kunitz (1). The preparation, as modified by Heppel and Hilmoo (2, 3), involves fractionation and crystallizat,ion from alcohol at temperatures as low as - 23” C, proccdurcts which arc inconvvcniont and difficult to reproduce. We have purified this yeast enzyme utilizing chromatographic tcchniqucs and have devised a simple procc\duro for crystallizing the enzyme from ammonium sulfate solution in a stable, homogcnoous form. We havo undertaken dctailtld inv&igations of ligand binding (4), ilIg2+ activation (5), and Ca2+ inhibition (6) of this crystalline 1 Paper periment
nmnber Station,
4797 of the Agricultural Purdue University.
yeast cnzymc. We report here the results our studies on its yuaternary structure, interaction with chelating agents, and ability to strongly bind Mg2+, as well as purification procedure. MATERIALS
@ 1972 hy Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Tris and ammonium sulfate were the “ultrapure” grade from Mann Hesearch Laboratories. CDTA2 and Elon were obtained from Aldrich Chemical Company; EGTA and imidazole were from Sigma Chemical Co. Eastman Organic Chemicals supplied PMSF. Fresh l-lb cakes of Budweiser baker’s yeast were obtained from a local bakery. Doubly deionized distilled water was used in all reagents employed except, for early steps in the purification. Glass vessels in contact with the prlrified enzyme were previously soaked in di* Nonstandard abbreviations include: CDTA, lrun.s-1,2-diamino-cyclohexane-.\~,N,N’,Nf-tetraacetic acid ; EGTA, ethyleneglycol-bis (p-aminoethylether).N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; Elan, p-methylaminophenol sulfate; PMHF, phenylmethane sulfonyl fluoride; dit,hizone, diphenylthiocarbazone.
Ex714
Copyright All rights
AND
of its its the
YEAST
PY ROPHOSPHATASE
chromic acid, soaked in soap solutions, and boiled in deionized wat.er, followed by prolonged soaking in several changes of deionized water. Metal ions were removed from buffers by dithizone extraction in CClr (7, 8). Residual CC14 was removed by ether extraction; residual ether was distilled off by stirring under a vacuum. Pyrophosphat,ase activity was measured as orthophosphate production by a modificat,ion of the Fiske-Subballow technique (9). The assay solution consisted of 1.0 ml of 2 my PP,, 2 mu RlgC12 ? and 0.1 mM F:GTA in 0.1 M Tris, pH 7.2. EGTA, which has an extremely weak affinity for iLIgl+ (lo), was inclrtded to complex any contaminating heavy metal ions; significantly higher rates were observed in the presence of IX+TA. After temperature equilibration at 3O”C, 0.01-0.1 unit of enzyme activity in 0.014.05 ml of 0.1 M Tris, 1~1% 7.2, was added to start the reaction. After 5 min at 30°C reaction was terminated by addition of 2.5 ml of “stop mix” made by mixing 4 vol of of water and 1 vol each of 5 N HzSOZ , 2.5’( (NH1)6M~i0X44H.L0 in water, and 3’2 NaHSOa-lV, I<:lon. Stop mix was freshly prepared daily, and stored on ice. Because of the small amount of protein present, centrifugation t,o remove precipitated protein from the acidified assay was unnecessary. rlfter color development for 15 min at 30°C the samples were read at 060 nm against a water Ihmk. Control samples (enzyme omitted) f standard amounts of Pi were included to ensure linearity between measured rate and enzyme concentration, A unit of pyrophosphatase activity is that, amount which hydrolyzes 1 pmole of pyrophosphate per minute under the above conditions. Protein concentration was measured by optical density at 280 nm Itsing Kunitz’ value of 1.45 for the A,,, of a 1.0 mg/ml solution of crystalline yrast pyrophosphatase (1). Fluorescence measurements and processing of fluorescence data were donr as previonsly described (4).
AND
BOUNI>
715
Mg2+
ml was added with stirring. After stirring the mixture for 1 hr and centrifuging as above, the supernatant fluid was carefully removed and discarded. The precipitate was dissolved in 300 ml of cold deionized water. /4cirZ preci$ation. Sufficient 1 iu MgCl~ was added to the dissolved precipitate (600SO0 ml) to make the MgCl~ concentration 50 mAI. Using a pH meter while stirring, the solution was t’itrated with 1 N acetic acid to pH 4.8 and stored for 12 hr after which the suspension was centrifuged as above, and t,hc prrcipit,ate discarded. The supcrnatant fract,ion was tit’ratcd to pH 6.0 with 1 s NaOH and dialyzed for at least 12 hr against 20 vol of dcionizcd water containing 0.1 ml1 EGTA. Batchwise
treatment
with DEAfi-cellulose.
The dialyzed acid-treated enzyme (approx 1.5 liters) was made 1 rnlr in JrgCIZ by addition of an appropriate volume of 1 ~1nlgci, , and the enzyme solution was t’itrated to pH 7.3 with solid Tris. Then Illamwx DEAEcellulose (Mann Research Laborat’orics) (500 g moist weight from Kichncr funnel, 200.ml volume) equilibratcbd with 0.01 M Tris, pH 7.3, w&h 1 rnlr 11gC12, was stirrcld into the enzyme solution. Aftw a few minutw the slurry was filtered on a Biichncr furmcl and washed with 500 ml watw. Under thrw corlditions the enzyme did not’ bind to the mat,rix, but approximately t’hrec-quarters of the protein was rrmowd. DEAE-c~~llulosc from other sources (see below\-)did bind the (‘nzymc under thrse conditions. First
DEAIkellulose
column
chromato~l-
The combined cluatc and washings (approx 2 lit’ers) were applied at a rapid RESULTS flop ratcb to a 7.S X ST-cm column of DEAEcellulose (Sigma Chemical Company) which I’ur$lication 0f theenzyme. Tolucnc~plasmolhas bwn c>quilibrated with 0.01 AI Tris, pH ysis and clxtraction of the yeast cells w~rc 7.3, containing 1 ml1 11gC12. After washing performed on 6 lb of compressed baker’s with ,500ml of the same buffer, the column J,cast exactly as dtwribcd by Kunitz (3). was clutcd nith a linear NaCl gradient’, O-O.3 From this point all operations were carricld 11,in 0.01 JJ Tris, pH 7.3, cont,aining 1 rn41 out, at. 3-5” C. 11gC12, using 2 liters of each buffer. The Awmonium s&jute jractionatiun. To the tubes of highest specific activity n-we comcombined filtrate and washings (approx 2 bined and dialyzed against lo+ 11 EDTA, lit(w) 0.295 g (NH&SO-l I\-as added per ml, Ivith stirring for at least 30 mins. The sus- then against 0.03 M imidazok, pH 6.8, POJ~pcGon was centrifuged at 15,000 !/ for 10 taining lo-” 31EDTA. Second DJJL41kellulose chromatoyraph y. min and the prccipitatr discarded. To the t;upc~rnat~nrit layor 0.160 g (NH&WI per The dialyzed enzyme was applied to a 4 X ?,aphy.
716
RIDLINGTON,
YANG,
23-cm column of DEAE-cellulose (Sigma) which had been equilibrated with 0.03 M imidazole, pH 6.8, containing 1O-4 RI EDTA, and after washing into the bed with 200 ml of the equilibrating buffer, the column was &ted with a NaCl gradient, O-O.2 11, in 0.03 31 imidazole, pH 6.5, containing 1O-4 JI EDTA, using 1 liter of each buffer. Considerable inact’ivc protein n-as elmed just ahead of and incompletely separated from the enzyme, which was retained on the column more strongly in these conditions t’han in the presence of 1\Ig2+, which it binds (4). Crgstallixation (adapted from Jacoby (11)). The protein, approximately 50% pure, was prccipit’ated by addition with stirring of solid ammonium sulfate to 90 % saturation (12), and centrifuged at’ ln,OOOg for 10 min. After carefully removing and discarding the supernatant fluid the pellet was extracted with unbuffered solutions containing lOPa31 EGTA which were successively 77, 7.3, 69, 65, and 62 % saturated with ammonium sulfate (12), 1 ml of solution per 5 mg of protein. After occasional stirring for 10 min the pellet was centrifuged at 15,000 ~1for 10 min. Each extracted eupcrnatant’ layer containing dissolved protein was carefully removed to a small stoppercd test’ tube, then the next extracting solution was added. Usually the pellet was completely solubilized by the 65 % saturated ammonium sulfate. Within a few days (in some cases,a few hours), crystals usually began to form in the extracts containing 73 and 69 % saturated ammonium sulfate. Under a light microscope, the usual crystals (Fig. la) have a superficial resemblance to the same enzyme crystallized from alcohol (1); occasionally a different crystal form was observed (Fig. lb). The specific activity of the crystals was usually approximately 6.50 units/mg. The crystals have been stored as the ammonium sulfate suspensionfor 3 years at 4” C without loss of activity. Several modifications of the procedure have been employed; early preparations were initiated by aut’olysis of dried yeast in bicarbonate buffer (l), and a variety of conditions have been employed for the chromatography. The only significant difference which we have observed in the crys-
AND
BUTLER
talline preparations obtained by somewhat different methods is that preparations obtained by t,oluene plasmolysis generally contain protease activity (see below) as a trace contaminant; protease activity was not, det’ccted in bicarbonate-autolyzcd preparations. Yield. The yield of activity is not presented at each step because large increases in total activity were often observed, presumably due to the presence of variable amounts of inhibitory materials in the early fractions. Final yields are usually in the range of 25,000 units (40 mg) of crystalline enzyme per 6 lb of moist yeast. Homogeneity. The chromatographically purified enzyme was not, homogcncous, as judged by its clution patt’ern which ovcrlapped inactive protein and by its specific activity of approximately 350 compared to the value of approximately 6;SOusually observed with crystalline enzyme. As shown by polyacrylamidc gel elrctrophoresis (Fig, 2) the chromatographically purified enzyme consiskd of two major elcctrophoretically separable components. Enzymcl assays on the gel (14) indicated that pyropbospha,tase activit,y is associakd with only the component with lower mobilit,y (upper band). A single crystallization appeared to rat,her effectively rcmovc the inactive contaminant (Fig. 2) ; no trace of the cont’aminant was apparent after crystallizat,ion. E’urt’hcr cvidence of t,he apparent’ homogeneity of the crystalline preparation is provided by ultracentrifugal studies described below. Molecular weight. Several preparat’ions of crystalline native enzyme subjected to sedimentation equilibrium analysis (15) gave in every case linear plots of In Z vs P, iridicat,ing a high degree of homogeneity (Fig. 3). The molecular weight of the enzyme, 71,000 f 2000, was independent of protein concentra.tion from 0.2 to 0.8 mg/ml. No evidence was obtained for association-dissociation of the native enzyme. The subunit molecular weight of the crystalline pyrophosphatase was determined by SDS gel clectrophorcsis (18) f dithiothreitol to reduce disulfide bonds and f PMSF to inactivat#e cont’aminat8ingproteases (19, 20) (see below). In each case a single protein band corresponding to molecular weight
YEAST
PYROPHOSPHATASE
AND
BOUND
&“+
FILG. 1. Yeast pyrophosphatase crystals. a, usual crystalline form (704X); b, occasional cry&a tlline form (704X). Crystallization was from ammonium sulfate solution as described in the text. al’P’ seen COIIF are
roxima,tely 34,SOOf on the gel, indicating
3000 (I;&. 4) was
that the enzyme :ists of two subunits of equal size which probably not held togthw by disulfide
boneIs. Electrophoresis in the prcwnce of S $1ur‘eain gels (23) and buffers instead of SDS also gave a single protein band, suggesting that the subunits aw similar, if not identical, in clh:trge as wcdl as mass.
Inievaciion
with Chelators: Enhancenlent
Fluorescen
ce
Cpon addition of certain chelators the intrinsic protein fluorcsccncc of ycwt pyrophosphatase (excit’ation at %SOnm, emieision at 345 nm) was obwrwd to bc consider;ably enhatwd (Fig. 5) ; in contrast, the prc$ein fluorcsc~ttca is yucnchcd on binding diva ,1ent
718
RIDLINGTON,
YANG,
FIG. 2. Gel electrophoresis of purified enzyme before and after crystallization. Analyses were conducted as described by Davis (13) using only the small-pore gel. Left, chromatographically purified yeast pyrophosphatase immediately before crystallization; right, crystalline enzyme.
AND
BUTLER
4.3
4.2
4.1 0
a
2
3
FIG. 4. Determination of subunit molecular weight by SDS gel electrophoresis. The technique was that of Weber and Osborne (IS), except that the ammonium persulfate concentration was halved, and the gel buffer was diluted 3-fold. The standard proteins are: BSA, bovine serum albumin, M, 67,000 (21); L-ADH, liver alcohol dehydrogenase, k’, 42,000 (22); G3-P, glyceraldehyde 3-phosphate dehydrogenase, M, 37,000 (23) ; and Cyt,. c, cytochrome c, M, 13,400 (24). X represents the mobility of the pyrophosphatase subunits.
49
50
51
r2bl12) FIG. 3. Molecular weight determinat,ion by sedimentation equilibrium. Crystalline pyrophosphatase, 0.3 mg/ml, was dialyzed for 24 hr at 10°C against 0.01 M Tris, pH 7.4, containing 0.1 M KCl. Centrifugation was for 24 hr at 12,590 rpm, at 4°C. Data obtained by absorption optics were analyzed by an automated computer technique (15) using 1.005 g cmM3 as the solvent density and 0.74 cm3g-1 as the partial specific volume (calculated (16) from the amino acid analysis (17)).
metal ions (4). The observations reported below suggestthat fluorescence enhancement by chelators is not simply due to complexation of contaminating metal ions which would otherwise bind to the enzyme and quench the fluorescence but is caused by direct interaction of chelators with the enzyme. The fluorescence enhancement due to chelators is readily and completely reversed by addition of excess divalent metal ions. This is most conveniently demonstrated with innocuous metal ions such as S?+, which does not activate the enzyme, and which in the absenceof chelator has no effect upon the protein fluorescence.3 The en3 J. W. Sperow observations.
and
L. G. Butler,
unpublished
YEAST
PYROPHOSPHATASE
AND
BOUND
719
MgZ+
-L~[EDTA] [EGTA].
M x 10' or [EOTA], hi x to'
FIG. 5. Fluorescence enhancement by chelating agents. Before use, crystalline pyrophosphatase was dialyzed 48 hr against two changes of 5 mM CDTA in 0.1 M Tris, pH 7.2, then for 24 hr against 0.4 i\l Tris, pH 7.4, with Chelex 100 (Calbiochem) added to the fluid outside the dialysis bag. To 1.0 ml of 0.4 M Tris, pH 7.4 equilibrated at 30°C in a 1 X l-cm fluorescence cell was added to 10-J of dialyzed enzyme; fluorescence was measured as previously described (4) using 280 nm as the excit,ation wavelength and 345 nm for emission. Chelators were added in 59.J portions approximately 3 min apart; chelator concentrations shown are the final, diluted concentrations. Two titrations with each chelator are shown: 0, 0, EDTA; A, X, EGTA.
$-b
-6
'
0
I I
'1
I 2
I
I
I
3
4
5
I/AF
hancement was observed even with enzyme preparat#ions from lvhich dissociable metal ions have been removed by thorough dialysis against chelators followed by dialysis against specially prepared metal-free buffer to remove the chelators. Nuorescence enhancement did not result in alteration of excitation or emission maxima. All chelators for \\-hich data are presented Jvere nonfluorescent as demonstrated by control titrations in the absence of enzyme. F’luorescence t,it,rations were carried out with a series of agents which bind divalent metal ions to Gdcly varying extents. A set of such titrations mith EDTA is presented in Fig. fja; for more accurate determination of apparent dissociation constants of the cnzymn-chclat’or binary complex thcx same data are plotted in linear form (26) in Fig. Ab. In this plot t’he ordinate intercept is - l/KYP. Observed values of K”o”” detcrmined in this manlier for a series of chelators as ~(~11 as observed degrecls of fluorescence
FIG. 6. Fluorescence titration with EDTA. The titration was conducted as described in the legend for Fig. 5 except that the Tris concentration was 0.24 M and the protein concentration was 1.0 X lo-& M before addition to the buffer. The different symbols represent four separate t,itrations: (a) conventional tit’rat,ion plot; (b) linear plot of same data. The ordinate intercept is -l/KD*pp accordingto the Benesi-Hildebrand equation (26).
enhancement at saturation are listed in Table I along with literat’urr values of chelatorMg2+ dissociation constants for purposes of comparison. The degree of fluoresccnccl pnbancemrnt at saturation of the protcin varies from 0 to 14 % depending upon t,hc nature of tho metal binding agent utilized (Table I). If the sole effect. of chelators is to bind contaminating dissociable metal ions, then all chelators which can form compltixcs with t,hc metal ions should produce the same degree of en-
720
RIDLINGTON, TABLE
YANG,
I
CHARACTERISTICS OF APPARENT CHEL.ITOR BINDING TO PYROPHOSPH.~T.LSE~ Chelator
Literature
Observed % Fluorescence enhancement at saturation
EDTA CDTA EGTA PPi Citrate
12 14 9 3 2
RD apparent (er~;m;~
2.1 4.8 4.1 6 1
x x X X x
lo-' lo-' lo+ 1o-3 lo-
KD
(Mgzc-chelator)
M M M M M
2.0 4.8 6.2 3.9 5.1
x x X X x
lo+ lo-" 10-O 1O-6 lo-
M M
M M M
a Fluorescence measurements were made as described in Fig. 5. Conditions were 0.24 M Tris (Cl), pH 7.4, 30°C. Literature values for dissociation constants of chelator-Mg2+ complexes were selected from values obtained under comparable conditions (10).
hancement. Instead, the differing degrees of fluorescence enhancement observed at saturation indicate that the chelators apparent,ly can bind directly to the enzyme, perturbing tryptophanyl residues to different extents depending on the structure of the chelators. The effect of a much more bulky chelaDor, 8-hydroxy-5quinolinesulfonic acid, could not be directly assessed due to the overlap between its absorption spectra and the emission spectra of the protein fluorescence. This chelator is itself fluorescent; no significant enhancement: quenching, or polarization of this fluorescence was observed in the prcsence of pyrophosphatase. Thus, there is no indication that this chelator can bind to the protein. Inactivation
in the Presence of Chelators.
Under certain conditions chelator-dependent irreversible inactivation of the enzyme was observed. This inactivation, which usually approximated first-order kinetics, was found to be rapid (half-life of 10 min under some conditions) in partially purified preparations, and slower but significant in highly purified crystalline samples prepared from toluene-plasmolyzed yeast. Examinat’ion by sedimentation equilibrium and gel
AND
BUTLER
electrophoresis of samples of crystalline enzyme which had been inactivated by exposure to 1 mM EDTA at pH 9.0 for 1 hr at 30” C revealed gross heterogeneity and extensive degradation of the protein (average M, = 10,000 f 5000). This suggested cont,amination by yeast protease, which other investigators have found to be a persistent impurity in otherwise highly purified preparations of yeast enzymes (19, 20, 27). Treatment with 1 mM PMSF for 1 hr at pH 7.4, 30’ C, inhibited proteolysis without affecting the pyrophosphatase activity. Preparations lacking protease activity as judged by nearly homogeneous patterns on gel electrophoresis after exposure t,o chelators in inactivating conditions (see below), nevertheless underwent a slow inactivation of the enzyme in the presence of chelators. Proteolyt’ic inactivation and degradation of yeast pyrophosphatase was observed only in the presence of chelators; in control experiments from which chelators were omitted the enzyme was quite stable. The structure of the native enzyme appears to be completely resistant’ to attack by yeast proteases in the absence of perturbation by chelators. Moreover, chelators can bind to the enzyme without inducing proteolysis; for example, in the conditions utilized for measuring chelator binding by fluorescence titration no inactivation was observed. Proteolytic degradation required not only chelator binding but certain other conditions of temperature and pH, etc. Optimum conditions for chelator-dependent inactivation were investigated with a partially purified preparation rich in protease activity. The chelator-dependent loss of pyrophosphatase activity was strongly influenced by temperature; under conditions in which 90 % of the activity was lost at 30” C there was no detectable loss of activity at 0” C. The inactivation rate increased rapidly with pH in the range pH 7-9. At pH 8.5 the observed rate of EDTA-dependent inactivation was 200-fold faster than at pH 7.2 in otherwise identical conditions. The inactivation rate also depended upon concentration of the chelator, as shown in Fig. 7. These data approximate a theoretical titration curve with KYp = 2 X lo+’ i\f for EDTA, similar to that observed fluoro-
YEAST
PYROPHOSPHATASE
AND BOUND
Mj$+ TABLE
M&J+
Sample 1 2 3 4 3
4
5
6
-LOO [EDTA]
Fro. 7. 1Late of inactivation as a function of EI1T.k concentration. Samples of partially purified rnzgmc (sp act 25) n-erc incnhated with variolls EDTA concentrations in 0.1 $1 Tris, pH 8.0. at 3O”C,. Portions (W ~1) were removed for enqqnatic assay at intervals, and the relative ratrs of inactivation were obtained from a firstorder plot of the primary data. The X’s represent observed rates of inactivation; the solid lint is a. theoretical curve for Ku = 2 X 10-j y.
metrically (Table I). Parallel, though lws extensive, cxpcriments with EGTA and CDTA revcalcd effects similar to thaw observed with EDT-4. A/eta1 Analyses The observed effects of chelators arc cw&tent’ with association of strongly bound m&al ions with t.he native enzyme. Duplicatcb IO- t,o IQ-mg samples of crystalline enzyme which had been dialyzed for GOhr at 10” C against t#hrcc changes of 0.1 ml1 EDTA, pH 7.4, wcrc analJ-zed for m&.l content b? spark-gap emission spectrometry. The following metals were undetcctablr: Zn, Sr, Ba, Fr, Ca,, Cr, Pb, I\In, Xi, Cd, and No. Al was present at lo\\- lewls, 0.03-0.07 molrs/;moln of enzyme (71,000 31,). The only other metal detected US Jig, which was present in significant’ but nonstoichiomc,tric amounts (maximum of 0% molt/ mok of c>nz?-me). Considering the long dialysis against EDTX and thr abscnw of other m&als, this Jig must have bwn vcq strongly bound to have bwn retainc~d with the protein even to this limited extent. Direct 31g2+ analysis by atomic absorption 5pwtromt+ry on scwral pwparations which had bren dialyzed for shortt>r timrs under mildw conditions, including a sample
CONTENT
721 II
OF PYROPHOSPFIATASLF
Specific
[Mg”f]/
activity
[protein]
[enzyme]
0.84 0.61 1.3 0.43
0.84 1 .O 1.8 1.6
650 395 470 175
D’k”+l/
_..~. caMg2+ analysis was by atomic absorption using a Perkins-Elmer spectrometer, Model 214. Samples were aspirated at a rate of 0.22-0.24 ml/min; at least 1.0 ml of sample cont,:rining approximately 5 X 10MG JI MgZc was analyeed. hlg*+ standards were made up ill buffer after it was shown that essentially identical values were obtained in buffer as in buffer containing 0.5 mg/ml of bovine serum albumin. Protein culcent ration was detrrmined by absorbancae at 280 nm (1); enzyme COW centration was determined k)?- the standard pyrellhosphatase assav. assuming a specifics activity of SO for fully aciive pure rnzl-me. Tmmediatel~ before allalJais, c’n~!vme was dialyzed at least 12 hr against 0.01 11 Tl:is, pH 7.4, from whic,h metal ions had been estract,ed as described ill 1lethods. In every case Mg?+ was uudetrctable ([Mg] < 0.2 PM) in the bnf’t’cr after dialysis. lIZvery sample except No. 3 had previouslv been exposed for several hljurs to 1;J jT.4 (c’ollc.elltratiolI > 0.1 mnl). Sample Xo. 2 had been partially inactivated by treatment with 0.1 rn~ ITh at ~11 9.0 for 5 hr at :IO”C. Sample X0. 4 was a c*ommercial preparation (Worthingtoll) which had becll stored froze11 for over 3 \-ears.
prepared commercially by independent techniques, revealed the presence of gwater amounts of ilIgZi (Table II). The stoichiometry is somewhat variable, but the results suggest the presence of at least’ one mole of Mg2+/‘mole of active enzyme; inactive prohein present in some casts contained relatively much lessIlIg2+. This l\Ig”+ associated nit’11 t,he enzq’m(’ must, have bwn strongly bound; in cont’rast the Rig”+ which activates the cnzymc~ is rclatiwly \vc&l~. bound (KD = 1.7 X 1OW 11(a)) and would haw bern rcmovcd by the prior dialysis of thwck samplw.
Direct correlation of catalytic activit’y with content of strongly bound 1\Ig2+ is complicated b>ythrb prcscnw of Mg’+ in t’he
722
RIDLINGTON,
YANG,
routine assay. Samples which have less than stoichiometric amounts of Mg*+ can have specific activities characteristic of fully active enzyme (Table II, sample I). This does not prove that the activit’y is independent of Mg2+ content; samples which contain less than stoichiomet’ric amounts of Mg*+ presumably rapidly bind Mg2+ when added to the assay mix. Catalytic activity was indirectly correlated with Mg*+ content using a partially purified pyrophosphatase preparation which contained active yeast protease. The preparation was treated with 10 rnl\r EDTA in 0.1 &I glycine, pH 9, at 45” C to rapidly inactivate the pyrophosphatase by removal of Mg2+ and proteolysis of the metal-free enzyme. At intervals throughout the inactivation portions were removed and immediately chilled to terminate any further Mg2+ release and proteolysis, and the nIg2+ already released was removed by dialysis. Calculated on the basis of catalytic activity, the initial preparation contained 0.97 Mg2+/mole of enzyme. The results, as shown in Icig. 8, indicate a rough correlation between h1g2+ content and catalytic activity remaining. Thus, in the presence of protease which rapidly inactivates the 1Ig*+-free pyrophos-
0
AND
BUTLER
phatase at high pH and temperature, a partially inactivated sample apparently consists of a mixture of native enzyme containing approximately 1 Mg*+ per mole, and and inactive digestion products which contain little or no nIg*+. Protease-free enzyme was treated with 5 rn>r EDTA at pH 9.0, 30” C, for 24 hr to remove Rig*+, and then the molecular weight (sedimentation equilibrium) was determined after dialysis at 5” C against 0.01 nr Tris, pH 7.4, containing 0.1 JI KCl. A control sample from which EDTA was omitted was fully active, and the molecular weight was 70,000. The EDTA-treated sample was heterogeneous, with an average molecular weight of 39,000 as estimated from the (nonlinear) plot)s of In Z vs 7,2.This is consistent with t’he presence, after extensive treatment with EDTA, of a mixture of 71,000 A/, enzyme dimers and 35,000 M, monomers. These experiments suggest that the strongly bound llIg2+ helps to maintain the enzyme in a dimeric form which is resistant to proteolytic digestion. A kinetic test was devised to more directly cxaminc the correlat’ion between R4g2+ content and catalytic act’ivity. A crystalline pyrophosphat,asc preparation essrntially free
I
I
I
I
I
I
I
I
I
I
5
IO
I5
20
25
30
35
40
45
50
MINUTES
OF
INACTIVATION
AT 45°C
FIG. 8. Correlation of loss of Mg2+ and activity. Partially purified enzyme (sp act 20), 144 mg protein, was incubated at 45°C in 20 ml of 0.1 M glycine, pH 9.0, containing 10 mM EDTA. At intervals 2-ml aliquota were removed, cooled in ice, and placed into dialysis bags which had been boiled in dilute EDTA solution. All samples were dialyzed for 12 hr at 5°C against each of the following buffers successively: 0.1 M Tris, pH 7.3; 0.01 M Tris, pH 7.3, containing 0.1 mM EDTA; 0.01 M Tris, pH 7.3, with Chelex 100 in the dialyzing buffer. After dialysis the samples were assayed for pyrophosphatase activity (dashed lined) by the standard assay and for Mg2+ content (solid line) by atomic absorption.
YEAST
PYROPHOSPHATASE
AND
BOUND
Mg2+
723
of yeast protease activity was incubated under conditions (5 ml1 EDTA, pH 9.0, 30” C) in which Mg2+ is rapidly removed, judging by the susceptibility to proteolysis in ot,hrr preparations on such t’reatment. In hhr normal assay this presumably i\Ig2+dcficicnt enzyme exhibited kinetics linear with timcx, as obscrvcd with native cnzymc (Fig. 9a). However, in assay conditions (0.3 rnlr JIgz+, 2 rnh1 ]‘I’,) where the concentration of free 11g2+ is very low, a definite lag was observed (Fig. 9b), indicating a noninstantaneous recovery of activity. Brief EDTA trcat,ment at pH 7.4 rather than pH 9.0 did not result in a lag in the low-11g2+ assay (Fig. 9c). These observations suggest that pyrophosphatase which does not contain strongly bound RIg2+ is inact,ive but can be rapidly converted to an active form at a rate which is dependent upon the conccntration of free Mg2+. Replacement of Mg2+ in the assay by eit.her lIn2+ or Zn2+ gave low levels of activity as previously observed with these ions as activat,ors (1); no increase in activity was observed with time as was found with _11g2+. It would appear that the only active enzyme in such conditions is that from which JIg2+ had not yet been removed. DISCUSSION
FIG. 9. Effect of assay conditions of Mg2+-deficient enzyme. Protease-free crystalline pyrophosphatase (0.03 mg) obtained by t’he bicarbonate extraction procedure was incubated at 30°C in 0.5 ml of 0.2 M Tris, pH 9.0, containing 5 mM EDTA. At various times small samples were removed and added to 10 ml of assay solution maint,ained at 30°C. One-milliliter portions were removed from this incubation at various times and assayed for orthophosphat,e product,. Figure 9a: standard assay conditions (2 rnM Mg*+, 2 mM PPi); -Obefore enzyme was exposed to EDTA at pH 9; ---O--: 30-min exposure; -a-70-min exposure. Figure 9b: “low Mgz+” assay (0.3 mM Mg2+, 2 mar PPi); -a--, before exposure to EDT.4 at pH 9; ---O-, 15-min exposure; -A-, 45-min exposure. Figure 9c: exposure to 1 rnM EDTA in 0.1 M Tris, pH 7.4 at 30°C. Activity was measured with the “low Mg*+” assajr (0.3 rnM Mg2+, 2 rnM
The original purification of yeast pyrophosphat,ase (1) has been supplanted by a variety of procedures (3, 28, 29); the principal advantage of this procedure is the simple method of crystallization and the stability of the crystals. The molecular weight’ of 71,000 as det’ermined by sediment’ation equilibrium is somewhat greater than the value of 63,000 obtained by Schachman using sedimentation velocity measurements (30). The presence of subunits of molecular weight of approximately 35,000 as found here and in Berlin (31) IS consistent with a 71,000 M, dimcr as the native enzyme. The dimeric sbructure of the molecule correlates well with the previous observations of 2.0 binding sites per 71,000 3, for divalent PPi). illiquots were assayed and 200-min exposure to EDTA; t,he same results as shown.
after 0-, lo-, GO-, all samples gave
RIDLINGTON,
724
YANG,
metal ions4 and for a Ca*+-pyrophosphate complex (4). Avaeva et al. had previously suggested that the enzyme consists of subunits (32). Molecular weights of 56,000, 36,000, and 12,000-17,000 for yeast pyrophosphatase and its subunits were obtained using enzyme chemically modified by maleic anhydride (3’2, 33). It is not known whether these preparations contained protease activity; the relationship of these results to our data is not clear. The fluorescence titrations indicat,e that chelators bind to the enzyme much less strongly than they bind Mg2+ (Table I) ; moreover, the relative strength of binding is different. EDTA binds t’o the enzyme more strongly than does CDTA, which has a somewhat greater affinity for free Mg2+ (10); perhaps this is a consequence of the greater bulkiness of CDTA and geometrical restrictions at the enzyme-binding site. The ext,remely weak binding of pyrophosphatase is in agreement, with direct binding and kinetic studies (4, 5). There appears to be a rough correlation between strength of binding of chelator to the enzyme and degree of enhancement of protein fluorescence (Table I>* We have previously demonstrated two distinct roles for ,1Ig*+ in this reaction: activation of the enzyme by binding free ptIg?+, convert’ing the enzyme to a form which can then bind substrate, which is a llIg2+ complex with pyrophosphate (4, 5). The present’ results suggest a t,hird role for Mg2+, maintenance of the stable native tertiary and quaternary structure of the enzyme. This apparent ‘(structural” Mg2+ is probably not the same as t,he “activating” RIg2+; for the lat.ter there are two binding sites per mole of dimer with K. = 1.7 X 10-j-S X 10P5 ~1 (4), but the structural RIg2+ may have only 1 site per dimer, and dissociation apparently does not occur to an appreciable extent in the absence of chelators. Zn2+, RIn2+, and Co2+ can replace nlg’+ in the first two roles, albeit with resulting lower activit’ies (1) ; kinetic measurements reported here using enzyme treated with EDTA t,o remove Mg2+ suggest that the structural Mg”+ cannot be 4 B. in(28).
Cooperman,
unpublished
results
cited
AND
BUTLER
replaced by Zn*+ or RIn*+ with retention of activit’y. In other experiments (not shown) it was found that incubation of the native enzyme with 1 rnnl Zn2+ or certain rare earths at’ pH 7.2, 30” C, resulted in rapid loss of activity; although Mg2+ and Zn2f analyses were not performed, these observations suggest that the strongly bound Mg2+ can be replaced, but with loss of activity. Nagnesium metalloenzymes are rare but not unknown (34). Scruttin and co-workers recently reported that pyruvate carboxylase from calf liver is a metalloenzyme which contains bound magnesium and bound manganese in a combined stoichiometry equivalent to the biotin content of the enzyme; the same enzyme prepared from livers of chickens raised on a manganese-deficient diet contains bound magnesium rather than bound manganese (35). 11g2+ is better known as a freely dissociable activating ion (36). As early as 1950, however, it was suggested that yea&-type inorganic pyrophosphatases probably contain mangesium (37). No pyrophosphatase has previously been shown to contain strongly bound metal ions, although a role for cobalt (38), manganese (39), or zinc (40) in maint’enance of activity of various bacterial pyrophosphatases has been demonstrated. Particularly interesting with respect to our findings is the latter case in ion while which Mg*+ is the activating Zn”+ is required to maintain the enzyme in an active form (40). Investigation of the yeast enzyme is continuing in our laboratory. ACKNOWLEDGMENTS We are grateful to Dr. B. Cooperman for communicating unpublished results and for suggestions of chromatographic conditions; to Dr. D. Filmer for ultracentrifugal analyses; to Drs. B. Vallee and H. Parker for metal analyses. This work was supported by Research Grants AM 12382 from the National Institllte of Arthritis and Metabolic Diseases and GB 8376 from the Nntional Science Folmdation. LGB is a recipient of a Research Career Development Award (GM 46404) from the U. S. Public Health Service. REFERENCES 1. KUNITZ, 2.
HEPPEL,
Hiol.
(1952). J. Gen. Physiol. 36,423. L. A., AND HILMOE, R. J. (1951) Chem. 192, 87. hf.
J.
YEAST
PYROPHOSPHATASE
RI. (1961) Arch. Biochem. Biophys. 3. KUNITZ, 92,270. L. Cr. J. 4. RIDLINGTON, J. W., AND BUTLER, Biol. Chem. in press. 5. MOE, 0. A., AND BUTLER, L. G. J.Biol.Chem., in press. 0. A., AND BUTLER, L.G. J.Biol.Chem., G. Mel:, in press. 11. E., LI, T. K., AND VALLEIJ, B. L. 7. I)RUM, (1969) Biochemistry 8,3783. Extraction of 8. STARY, J. (1964) in The Solvent lMetal Chelates (Irving, H., ed.), p. 136, Macmillan Company, New York. C. H., AND SUBBARO~, Y. H. (1925) 9. FISKE, J. Biol. Chem. 66,375. G.: AND MARTELL, A. E. (1964) in 10. SILLEN,L. Stability Constants of Met,al-Ion Complexes, p. 697, The Chemical Society, London. Il. JXOBY, W. B. (1968) Anal. Biochem. 26, 295. E. A., GUBLER, C. J., AND KGBY, 12. NOLTMANN, S. A. (1961) J. Biol. Chem. 236, 1225. B. J. (1964) Ann. N. Y. Acad. Sci. 121, 13. DAVIS! 404. A. (1967) J. Biol. 14. TONO, H., AND KORNBERG, Chew 242, 2375. D. L. Submitted to Anal. Biochem. 15. FILMER, (1957) in Methods in HI. K. 16. BCH.4CHMAN, Enzymology (Colowick, S. P , and Kaplan, N. O., eds.), Vo!. 4, p. 70, Academic Press, New York. W. (1952) J. Amer. chef% k’c. 17. HAUSMANN, 74,313s. I., AND OSBORN, M. (1969) J. Biol. 18. WEBER, Chem. 244,4406. J. R. (1970) Biochem. Biophys. Res. 19. PRINGLR, Commun. 39, 46. 20. RUST~M,Y.M.,MASSARO, E. J., ANDBARNARD, E. A. (1971) Biochemistry 10,3509. 21. LOEB, G. I., AND SCHERAGA, H. A. (1956) J. Phys. Chem. 60, 1633. 22. LI, T. K., AND VALLEE, B. L. (1964) Biochemistry 3,869. 23. HARRIS, J. L., AND PERHaM, R. ?;. (1965) J. Mol. Biol. 13,876.
AND
BOUND
Mg*’
725
24. EHRENRERG, A. (1957) Acla Chem. Stand. 11, 1257. 25. JOVIN, T., CHRliMBACH, 9., ‘4ND NAUGHTON, RI. A. (1964) Anal. Biochem. 9, 351. 26. BLNESI, H. A., AND HILDILBRAND, J. H. (1949) J. Amer. Chem. Sot. ‘71, 2703. 27. FOSSET, M., MUIR, I,. W., XEILSEN, L. D., AND FISCHER, E. H. (1971) Biochemistry 10, 4105. 28. BUTLRR, L. G. (1971), in The Enzymes (Boyer, P. D., ed.), 3rd edition, Vol. IV, p. 529, Academic Press, New York. 29. NEGI, T., AND IRIB, ill. (1971) J. Biochem. 70, 165. 30. SCHACHMAN, H. K. (1952) J. Gen. Physiol. 36, 451. 31. HOFM~NN, E., AND BOEHME, H.-J. (19il) Fed. Eur. Biochem. Sot. Left. 17, 1. 32. Av.4w.4, S. M., BRAGA, E. A., AND EGOROV, *4. ill. (1968) Biofizika 13,1126. 33. A$v.4cv~, S. M. (1970) Proceeding of the Fifth Anniversary Meeting of the Laboratory of Bioorganic Chemistry, Lomonosov State University, Moscow, December 1970, p. 211. 34. KUBO~~ITZ, F., AND LUTTGENS, W. (1940) Biochena. 2. 307, 170. 35. SCRUTTON, M. C., GRIMINGER, P., AND WALLACB, J. C. (1972) J. Biol. Chem. 247, 3305. 36. VALJ,EL, B. L. (1960) in, The Enzymes (Boyer, P. D., L.IRDP, H. A., .~ND MYRBBCI~, K., eds.), 2nd edition, Vol. 3, p. 225, Academic Press, New York. 37. ROCHE, J. (1950) in The Enzymes (Sumner, J. B., and MYRB&X, K., eds.), 1st edition, Vol. I, Part I, p. 493, Academic Press, New York. 38. OGINSKT, E. L., AND RUMBAUGH, H. L. (1955) J. Bac(erio1. 70,92. 39. TONO, II.? .~ND KORNBERC,, A. (1967) J. Bid. Chenz. 242,2375. 40. KLEMME, J.-H., KLEEME, B., AND GEST, H. (1971) J. Bacleriol. 108, 1122.
OF
BIOCHEMISTRY
.\ND
Yeast IV. Purification,
Quaternary
inorganic
of Biochemisky,
714-725
(1972)
Pyrophosphatase
Structure,
J. W. RIDLINGTON, Department
163,
HIOPHYSICS
and Evidence
Y. YANG, Purdue Received
AND
Ilniversily, July
for Strongly
Bound Mg’+’
L. G. BUTLER Lafayette,
Indiana
g7907
5, 1972
Yeast inorganic pyrophosphatase has been purified to apparent homogeneity by salt fractionation and chromatographic techniques avoiding the use of organic solvents. The enzyme is obtained in stable crystalline form. The molecular weight of the native eueyme is 71,000 & 2000, and it consists of two subunits of similar, if not identical, mass and charge. The enzyme as isolated contains approximately 1 mole of strongly bound Mg2+ per mole of active dimer; certain chelating agents cause an enhancement of the intrinsic protein fluorescence, presumably by complexing with this Mgz+ while it, remains bound to the enzyme. The Mg2+ can be partially removed by chelators under specified conditious; the metal-free enzyme appears to be inactive, and is much more susceptible to proteolytic digestion and dissociation to monomers than is the native enzyme. The activity of met,al-free enzyme may be rapidly restored suggest that Zn2+ or other ions may replace the in the presence of Mg 2+; the results strongly bound M$+, with consequent loss of activity.
The inorganic pyrophosphatasc (pyrophosphatase phosphohydrolasc EC 3.6.1.1) reaction is perhaps the simplest system, in terms of the chemical structures involved, for enzymatic utilization of physiological polyphosphates, and is, therefore, an attractive model for mccha,nistic studios. For many years the only available pure (crystalline) pyrophosphatase was that obtained from baker’s yeast using the procedura describrd by Kunitz (1). The preparation, as modified by Heppel and Hilmoo (2, 3), involves fractionation and crystallizat,ion from alcohol at temperatures as low as - 23” C, proccdurcts which arc inconvvcniont and difficult to reproduce. We have purified this yeast enzyme utilizing chromatographic tcchniqucs and have devised a simple procc\duro for crystallizing the enzyme from ammonium sulfate solution in a stable, homogcnoous form. We havo undertaken dctailtld inv&igations of ligand binding (4), ilIg2+ activation (5), and Ca2+ inhibition (6) of this crystalline 1 Paper periment
nmnber Station,
4797 of the Agricultural Purdue University.
yeast cnzymc. We report here the results our studies on its yuaternary structure, interaction with chelating agents, and ability to strongly bind Mg2+, as well as purification procedure. MATERIALS
@ 1972 hy Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Tris and ammonium sulfate were the “ultrapure” grade from Mann Hesearch Laboratories. CDTA2 and Elon were obtained from Aldrich Chemical Company; EGTA and imidazole were from Sigma Chemical Co. Eastman Organic Chemicals supplied PMSF. Fresh l-lb cakes of Budweiser baker’s yeast were obtained from a local bakery. Doubly deionized distilled water was used in all reagents employed except, for early steps in the purification. Glass vessels in contact with the prlrified enzyme were previously soaked in di* Nonstandard abbreviations include: CDTA, lrun.s-1,2-diamino-cyclohexane-.\~,N,N’,Nf-tetraacetic acid ; EGTA, ethyleneglycol-bis (p-aminoethylether).N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; Elan, p-methylaminophenol sulfate; PMHF, phenylmethane sulfonyl fluoride; dit,hizone, diphenylthiocarbazone.
Ex714
Copyright All rights
AND
of its its the
YEAST
PY ROPHOSPHATASE
chromic acid, soaked in soap solutions, and boiled in deionized wat.er, followed by prolonged soaking in several changes of deionized water. Metal ions were removed from buffers by dithizone extraction in CClr (7, 8). Residual CC14 was removed by ether extraction; residual ether was distilled off by stirring under a vacuum. Pyrophosphat,ase activity was measured as orthophosphate production by a modificat,ion of the Fiske-Subballow technique (9). The assay solution consisted of 1.0 ml of 2 my PP,, 2 mu RlgC12 ? and 0.1 mM F:GTA in 0.1 M Tris, pH 7.2. EGTA, which has an extremely weak affinity for iLIgl+ (lo), was inclrtded to complex any contaminating heavy metal ions; significantly higher rates were observed in the presence of IX+TA. After temperature equilibration at 3O”C, 0.01-0.1 unit of enzyme activity in 0.014.05 ml of 0.1 M Tris, 1~1% 7.2, was added to start the reaction. After 5 min at 30°C reaction was terminated by addition of 2.5 ml of “stop mix” made by mixing 4 vol of of water and 1 vol each of 5 N HzSOZ , 2.5’( (NH1)6M~i0X44H.L0 in water, and 3’2 NaHSOa-lV, I<:lon. Stop mix was freshly prepared daily, and stored on ice. Because of the small amount of protein present, centrifugation t,o remove precipitated protein from the acidified assay was unnecessary. rlfter color development for 15 min at 30°C the samples were read at 060 nm against a water Ihmk. Control samples (enzyme omitted) f standard amounts of Pi were included to ensure linearity between measured rate and enzyme concentration, A unit of pyrophosphatase activity is that, amount which hydrolyzes 1 pmole of pyrophosphate per minute under the above conditions. Protein concentration was measured by optical density at 280 nm Itsing Kunitz’ value of 1.45 for the A,,, of a 1.0 mg/ml solution of crystalline yrast pyrophosphatase (1). Fluorescence measurements and processing of fluorescence data were donr as previonsly described (4).
AND
BOUNI>
715
Mg2+
ml was added with stirring. After stirring the mixture for 1 hr and centrifuging as above, the supernatant fluid was carefully removed and discarded. The precipitate was dissolved in 300 ml of cold deionized water. /4cirZ preci$ation. Sufficient 1 iu MgCl~ was added to the dissolved precipitate (600SO0 ml) to make the MgCl~ concentration 50 mAI. Using a pH meter while stirring, the solution was t’itrated with 1 N acetic acid to pH 4.8 and stored for 12 hr after which the suspension was centrifuged as above, and t,hc prrcipit,ate discarded. The supcrnatant fract,ion was tit’ratcd to pH 6.0 with 1 s NaOH and dialyzed for at least 12 hr against 20 vol of dcionizcd water containing 0.1 ml1 EGTA. Batchwise
treatment
with DEAfi-cellulose.
The dialyzed acid-treated enzyme (approx 1.5 liters) was made 1 rnlr in JrgCIZ by addition of an appropriate volume of 1 ~1nlgci, , and the enzyme solution was t’itrated to pH 7.3 with solid Tris. Then Illamwx DEAEcellulose (Mann Research Laborat’orics) (500 g moist weight from Kichncr funnel, 200.ml volume) equilibratcbd with 0.01 M Tris, pH 7.3, w&h 1 rnlr 11gC12, was stirrcld into the enzyme solution. Aftw a few minutw the slurry was filtered on a Biichncr furmcl and washed with 500 ml watw. Under thrw corlditions the enzyme did not’ bind to the mat,rix, but approximately t’hrec-quarters of the protein was rrmowd. DEAE-c~~llulosc from other sources (see below\-)did bind the (‘nzymc under thrse conditions. First
DEAIkellulose
column
chromato~l-
The combined cluatc and washings (approx 2 lit’ers) were applied at a rapid RESULTS flop ratcb to a 7.S X ST-cm column of DEAEcellulose (Sigma Chemical Company) which I’ur$lication 0f theenzyme. Tolucnc~plasmolhas bwn c>quilibrated with 0.01 AI Tris, pH ysis and clxtraction of the yeast cells w~rc 7.3, containing 1 ml1 11gC12. After washing performed on 6 lb of compressed baker’s with ,500ml of the same buffer, the column J,cast exactly as dtwribcd by Kunitz (3). was clutcd nith a linear NaCl gradient’, O-O.3 From this point all operations were carricld 11,in 0.01 JJ Tris, pH 7.3, cont,aining 1 rn41 out, at. 3-5” C. 11gC12, using 2 liters of each buffer. The Awmonium s&jute jractionatiun. To the tubes of highest specific activity n-we comcombined filtrate and washings (approx 2 bined and dialyzed against lo+ 11 EDTA, lit(w) 0.295 g (NH&SO-l I\-as added per ml, Ivith stirring for at least 30 mins. The sus- then against 0.03 M imidazok, pH 6.8, POJ~pcGon was centrifuged at 15,000 !/ for 10 taining lo-” 31EDTA. Second DJJL41kellulose chromatoyraph y. min and the prccipitatr discarded. To the t;upc~rnat~nrit layor 0.160 g (NH&WI per The dialyzed enzyme was applied to a 4 X ?,aphy.
716
RIDLINGTON,
YANG,
23-cm column of DEAE-cellulose (Sigma) which had been equilibrated with 0.03 M imidazole, pH 6.8, containing 1O-4 RI EDTA, and after washing into the bed with 200 ml of the equilibrating buffer, the column was &ted with a NaCl gradient, O-O.2 11, in 0.03 31 imidazole, pH 6.5, containing 1O-4 JI EDTA, using 1 liter of each buffer. Considerable inact’ivc protein n-as elmed just ahead of and incompletely separated from the enzyme, which was retained on the column more strongly in these conditions t’han in the presence of 1\Ig2+, which it binds (4). Crgstallixation (adapted from Jacoby (11)). The protein, approximately 50% pure, was prccipit’ated by addition with stirring of solid ammonium sulfate to 90 % saturation (12), and centrifuged at’ ln,OOOg for 10 min. After carefully removing and discarding the supernatant fluid the pellet was extracted with unbuffered solutions containing lOPa31 EGTA which were successively 77, 7.3, 69, 65, and 62 % saturated with ammonium sulfate (12), 1 ml of solution per 5 mg of protein. After occasional stirring for 10 min the pellet was centrifuged at 15,000 ~1for 10 min. Each extracted eupcrnatant’ layer containing dissolved protein was carefully removed to a small stoppercd test’ tube, then the next extracting solution was added. Usually the pellet was completely solubilized by the 65 % saturated ammonium sulfate. Within a few days (in some cases,a few hours), crystals usually began to form in the extracts containing 73 and 69 % saturated ammonium sulfate. Under a light microscope, the usual crystals (Fig. la) have a superficial resemblance to the same enzyme crystallized from alcohol (1); occasionally a different crystal form was observed (Fig. lb). The specific activity of the crystals was usually approximately 6.50 units/mg. The crystals have been stored as the ammonium sulfate suspensionfor 3 years at 4” C without loss of activity. Several modifications of the procedure have been employed; early preparations were initiated by aut’olysis of dried yeast in bicarbonate buffer (l), and a variety of conditions have been employed for the chromatography. The only significant difference which we have observed in the crys-
AND
BUTLER
talline preparations obtained by somewhat different methods is that preparations obtained by t,oluene plasmolysis generally contain protease activity (see below) as a trace contaminant; protease activity was not, det’ccted in bicarbonate-autolyzcd preparations. Yield. The yield of activity is not presented at each step because large increases in total activity were often observed, presumably due to the presence of variable amounts of inhibitory materials in the early fractions. Final yields are usually in the range of 25,000 units (40 mg) of crystalline enzyme per 6 lb of moist yeast. Homogeneity. The chromatographically purified enzyme was not, homogcncous, as judged by its clution patt’ern which ovcrlapped inactive protein and by its specific activity of approximately 350 compared to the value of approximately 6;SOusually observed with crystalline enzyme. As shown by polyacrylamidc gel elrctrophoresis (Fig, 2) the chromatographically purified enzyme consiskd of two major elcctrophoretically separable components. Enzymcl assays on the gel (14) indicated that pyropbospha,tase activit,y is associakd with only the component with lower mobilit,y (upper band). A single crystallization appeared to rat,her effectively rcmovc the inactive contaminant (Fig. 2) ; no trace of the cont’aminant was apparent after crystallizat,ion. E’urt’hcr cvidence of t,he apparent’ homogeneity of the crystalline preparation is provided by ultracentrifugal studies described below. Molecular weight. Several preparat’ions of crystalline native enzyme subjected to sedimentation equilibrium analysis (15) gave in every case linear plots of In Z vs P, iridicat,ing a high degree of homogeneity (Fig. 3). The molecular weight of the enzyme, 71,000 f 2000, was independent of protein concentra.tion from 0.2 to 0.8 mg/ml. No evidence was obtained for association-dissociation of the native enzyme. The subunit molecular weight of the crystalline pyrophosphatase was determined by SDS gel clectrophorcsis (18) f dithiothreitol to reduce disulfide bonds and f PMSF to inactivat#e cont’aminat8ingproteases (19, 20) (see below). In each case a single protein band corresponding to molecular weight
YEAST
PYROPHOSPHATASE
AND
BOUND
&“+
FILG. 1. Yeast pyrophosphatase crystals. a, usual crystalline form (704X); b, occasional cry&a tlline form (704X). Crystallization was from ammonium sulfate solution as described in the text. al’P’ seen COIIF are
roxima,tely 34,SOOf on the gel, indicating
3000 (I;&. 4) was
that the enzyme :ists of two subunits of equal size which probably not held togthw by disulfide
boneIs. Electrophoresis in the prcwnce of S $1ur‘eain gels (23) and buffers instead of SDS also gave a single protein band, suggesting that the subunits aw similar, if not identical, in clh:trge as wcdl as mass.
Inievaciion
with Chelators: Enhancenlent
Fluorescen
ce
Cpon addition of certain chelators the intrinsic protein fluorcsccncc of ycwt pyrophosphatase (excit’ation at %SOnm, emieision at 345 nm) was obwrwd to bc consider;ably enhatwd (Fig. 5) ; in contrast, the prc$ein fluorcsc~ttca is yucnchcd on binding diva ,1ent
718
RIDLINGTON,
YANG,
FIG. 2. Gel electrophoresis of purified enzyme before and after crystallization. Analyses were conducted as described by Davis (13) using only the small-pore gel. Left, chromatographically purified yeast pyrophosphatase immediately before crystallization; right, crystalline enzyme.
AND
BUTLER
4.3
4.2
4.1 0
a
2
3
FIG. 4. Determination of subunit molecular weight by SDS gel electrophoresis. The technique was that of Weber and Osborne (IS), except that the ammonium persulfate concentration was halved, and the gel buffer was diluted 3-fold. The standard proteins are: BSA, bovine serum albumin, M, 67,000 (21); L-ADH, liver alcohol dehydrogenase, k’, 42,000 (22); G3-P, glyceraldehyde 3-phosphate dehydrogenase, M, 37,000 (23) ; and Cyt,. c, cytochrome c, M, 13,400 (24). X represents the mobility of the pyrophosphatase subunits.
49
50
51
r2bl12) FIG. 3. Molecular weight determinat,ion by sedimentation equilibrium. Crystalline pyrophosphatase, 0.3 mg/ml, was dialyzed for 24 hr at 10°C against 0.01 M Tris, pH 7.4, containing 0.1 M KCl. Centrifugation was for 24 hr at 12,590 rpm, at 4°C. Data obtained by absorption optics were analyzed by an automated computer technique (15) using 1.005 g cmM3 as the solvent density and 0.74 cm3g-1 as the partial specific volume (calculated (16) from the amino acid analysis (17)).
metal ions (4). The observations reported below suggestthat fluorescence enhancement by chelators is not simply due to complexation of contaminating metal ions which would otherwise bind to the enzyme and quench the fluorescence but is caused by direct interaction of chelators with the enzyme. The fluorescence enhancement due to chelators is readily and completely reversed by addition of excess divalent metal ions. This is most conveniently demonstrated with innocuous metal ions such as S?+, which does not activate the enzyme, and which in the absenceof chelator has no effect upon the protein fluorescence.3 The en3 J. W. Sperow observations.
and
L. G. Butler,
unpublished
YEAST
PYROPHOSPHATASE
AND
BOUND
719
MgZ+
-L~[EDTA] [EGTA].
M x 10' or [EOTA], hi x to'
FIG. 5. Fluorescence enhancement by chelating agents. Before use, crystalline pyrophosphatase was dialyzed 48 hr against two changes of 5 mM CDTA in 0.1 M Tris, pH 7.2, then for 24 hr against 0.4 i\l Tris, pH 7.4, with Chelex 100 (Calbiochem) added to the fluid outside the dialysis bag. To 1.0 ml of 0.4 M Tris, pH 7.4 equilibrated at 30°C in a 1 X l-cm fluorescence cell was added to 10-J of dialyzed enzyme; fluorescence was measured as previously described (4) using 280 nm as the excit,ation wavelength and 345 nm for emission. Chelators were added in 59.J portions approximately 3 min apart; chelator concentrations shown are the final, diluted concentrations. Two titrations with each chelator are shown: 0, 0, EDTA; A, X, EGTA.
$-b
-6
'
0
I I
'1
I 2
I
I
I
3
4
5
I/AF
hancement was observed even with enzyme preparat#ions from lvhich dissociable metal ions have been removed by thorough dialysis against chelators followed by dialysis against specially prepared metal-free buffer to remove the chelators. Nuorescence enhancement did not result in alteration of excitation or emission maxima. All chelators for \\-hich data are presented Jvere nonfluorescent as demonstrated by control titrations in the absence of enzyme. F’luorescence t,it,rations were carried out with a series of agents which bind divalent metal ions to Gdcly varying extents. A set of such titrations mith EDTA is presented in Fig. fja; for more accurate determination of apparent dissociation constants of the cnzymn-chclat’or binary complex thcx same data are plotted in linear form (26) in Fig. Ab. In this plot t’he ordinate intercept is - l/KYP. Observed values of K”o”” detcrmined in this manlier for a series of chelators as ~(~11 as observed degrecls of fluorescence
FIG. 6. Fluorescence titration with EDTA. The titration was conducted as described in the legend for Fig. 5 except that the Tris concentration was 0.24 M and the protein concentration was 1.0 X lo-& M before addition to the buffer. The different symbols represent four separate t,itrations: (a) conventional tit’rat,ion plot; (b) linear plot of same data. The ordinate intercept is -l/KD*pp accordingto the Benesi-Hildebrand equation (26).
enhancement at saturation are listed in Table I along with literat’urr values of chelatorMg2+ dissociation constants for purposes of comparison. The degree of fluoresccnccl pnbancemrnt at saturation of the protcin varies from 0 to 14 % depending upon t,hc nature of tho metal binding agent utilized (Table I). If the sole effect. of chelators is to bind contaminating dissociable metal ions, then all chelators which can form compltixcs with t,hc metal ions should produce the same degree of en-
720
RIDLINGTON, TABLE
YANG,
I
CHARACTERISTICS OF APPARENT CHEL.ITOR BINDING TO PYROPHOSPH.~T.LSE~ Chelator
Literature
Observed % Fluorescence enhancement at saturation
EDTA CDTA EGTA PPi Citrate
12 14 9 3 2
RD apparent (er~;m;~
2.1 4.8 4.1 6 1
x x X X x
lo-' lo-' lo+ 1o-3 lo-
KD
(Mgzc-chelator)
M M M M M
2.0 4.8 6.2 3.9 5.1
x x X X x
lo+ lo-" 10-O 1O-6 lo-
M M
M M M
a Fluorescence measurements were made as described in Fig. 5. Conditions were 0.24 M Tris (Cl), pH 7.4, 30°C. Literature values for dissociation constants of chelator-Mg2+ complexes were selected from values obtained under comparable conditions (10).
hancement. Instead, the differing degrees of fluorescence enhancement observed at saturation indicate that the chelators apparent,ly can bind directly to the enzyme, perturbing tryptophanyl residues to different extents depending on the structure of the chelators. The effect of a much more bulky chelaDor, 8-hydroxy-5quinolinesulfonic acid, could not be directly assessed due to the overlap between its absorption spectra and the emission spectra of the protein fluorescence. This chelator is itself fluorescent; no significant enhancement: quenching, or polarization of this fluorescence was observed in the prcsence of pyrophosphatase. Thus, there is no indication that this chelator can bind to the protein. Inactivation
in the Presence of Chelators.
Under certain conditions chelator-dependent irreversible inactivation of the enzyme was observed. This inactivation, which usually approximated first-order kinetics, was found to be rapid (half-life of 10 min under some conditions) in partially purified preparations, and slower but significant in highly purified crystalline samples prepared from toluene-plasmolyzed yeast. Examinat’ion by sedimentation equilibrium and gel
AND
BUTLER
electrophoresis of samples of crystalline enzyme which had been inactivated by exposure to 1 mM EDTA at pH 9.0 for 1 hr at 30” C revealed gross heterogeneity and extensive degradation of the protein (average M, = 10,000 f 5000). This suggested cont,amination by yeast protease, which other investigators have found to be a persistent impurity in otherwise highly purified preparations of yeast enzymes (19, 20, 27). Treatment with 1 mM PMSF for 1 hr at pH 7.4, 30’ C, inhibited proteolysis without affecting the pyrophosphatase activity. Preparations lacking protease activity as judged by nearly homogeneous patterns on gel electrophoresis after exposure t,o chelators in inactivating conditions (see below), nevertheless underwent a slow inactivation of the enzyme in the presence of chelators. Proteolyt’ic inactivation and degradation of yeast pyrophosphatase was observed only in the presence of chelators; in control experiments from which chelators were omitted the enzyme was quite stable. The structure of the native enzyme appears to be completely resistant’ to attack by yeast proteases in the absence of perturbation by chelators. Moreover, chelators can bind to the enzyme without inducing proteolysis; for example, in the conditions utilized for measuring chelator binding by fluorescence titration no inactivation was observed. Proteolytic degradation required not only chelator binding but certain other conditions of temperature and pH, etc. Optimum conditions for chelator-dependent inactivation were investigated with a partially purified preparation rich in protease activity. The chelator-dependent loss of pyrophosphatase activity was strongly influenced by temperature; under conditions in which 90 % of the activity was lost at 30” C there was no detectable loss of activity at 0” C. The inactivation rate increased rapidly with pH in the range pH 7-9. At pH 8.5 the observed rate of EDTA-dependent inactivation was 200-fold faster than at pH 7.2 in otherwise identical conditions. The inactivation rate also depended upon concentration of the chelator, as shown in Fig. 7. These data approximate a theoretical titration curve with KYp = 2 X lo+’ i\f for EDTA, similar to that observed fluoro-
YEAST
PYROPHOSPHATASE
AND BOUND
Mj$+ TABLE
M&J+
Sample 1 2 3 4 3
4
5
6
-LOO [EDTA]
Fro. 7. 1Late of inactivation as a function of EI1T.k concentration. Samples of partially purified rnzgmc (sp act 25) n-erc incnhated with variolls EDTA concentrations in 0.1 $1 Tris, pH 8.0. at 3O”C,. Portions (W ~1) were removed for enqqnatic assay at intervals, and the relative ratrs of inactivation were obtained from a firstorder plot of the primary data. The X’s represent observed rates of inactivation; the solid lint is a. theoretical curve for Ku = 2 X 10-j y.
metrically (Table I). Parallel, though lws extensive, cxpcriments with EGTA and CDTA revcalcd effects similar to thaw observed with EDT-4. A/eta1 Analyses The observed effects of chelators arc cw&tent’ with association of strongly bound m&al ions with t.he native enzyme. Duplicatcb IO- t,o IQ-mg samples of crystalline enzyme which had been dialyzed for GOhr at 10” C against t#hrcc changes of 0.1 ml1 EDTA, pH 7.4, wcrc analJ-zed for m&.l content b? spark-gap emission spectrometry. The following metals were undetcctablr: Zn, Sr, Ba, Fr, Ca,, Cr, Pb, I\In, Xi, Cd, and No. Al was present at lo\\- lewls, 0.03-0.07 molrs/;moln of enzyme (71,000 31,). The only other metal detected US Jig, which was present in significant’ but nonstoichiomc,tric amounts (maximum of 0% molt/ mok of c>nz?-me). Considering the long dialysis against EDTX and thr abscnw of other m&als, this Jig must have bwn vcq strongly bound to have bwn retainc~d with the protein even to this limited extent. Direct 31g2+ analysis by atomic absorption 5pwtromt+ry on scwral pwparations which had bren dialyzed for shortt>r timrs under mildw conditions, including a sample
CONTENT
721 II
OF PYROPHOSPFIATASLF
Specific
[Mg”f]/
activity
[protein]
[enzyme]
0.84 0.61 1.3 0.43
0.84 1 .O 1.8 1.6
650 395 470 175
D’k”+l/
_..~. caMg2+ analysis was by atomic absorption using a Perkins-Elmer spectrometer, Model 214. Samples were aspirated at a rate of 0.22-0.24 ml/min; at least 1.0 ml of sample cont,:rining approximately 5 X 10MG JI MgZc was analyeed. hlg*+ standards were made up ill buffer after it was shown that essentially identical values were obtained in buffer as in buffer containing 0.5 mg/ml of bovine serum albumin. Protein culcent ration was detrrmined by absorbancae at 280 nm (1); enzyme COW centration was determined k)?- the standard pyrellhosphatase assav. assuming a specifics activity of SO for fully aciive pure rnzl-me. Tmmediatel~ before allalJais, c’n~!vme was dialyzed at least 12 hr against 0.01 11 Tl:is, pH 7.4, from whic,h metal ions had been estract,ed as described ill 1lethods. In every case Mg?+ was uudetrctable ([Mg] < 0.2 PM) in the bnf’t’cr after dialysis. lIZvery sample except No. 3 had previouslv been exposed for several hljurs to 1;J jT.4 (c’ollc.elltratiolI > 0.1 mnl). Sample Xo. 2 had been partially inactivated by treatment with 0.1 rn~ ITh at ~11 9.0 for 5 hr at :IO”C. Sample X0. 4 was a c*ommercial preparation (Worthingtoll) which had becll stored froze11 for over 3 \-ears.
prepared commercially by independent techniques, revealed the presence of gwater amounts of ilIgZi (Table II). The stoichiometry is somewhat variable, but the results suggest the presence of at least’ one mole of Mg2+/‘mole of active enzyme; inactive prohein present in some casts contained relatively much lessIlIg2+. This l\Ig”+ associated nit’11 t,he enzq’m(’ must, have bwn strongly bound; in cont’rast the Rig”+ which activates the cnzymc~ is rclatiwly \vc&l~. bound (KD = 1.7 X 1OW 11(a)) and would haw bern rcmovcd by the prior dialysis of thwck samplw.
Direct correlation of catalytic activit’y with content of strongly bound 1\Ig2+ is complicated b>ythrb prcscnw of Mg’+ in t’he
722
RIDLINGTON,
YANG,
routine assay. Samples which have less than stoichiometric amounts of Mg*+ can have specific activities characteristic of fully active enzyme (Table II, sample I). This does not prove that the activit’y is independent of Mg2+ content; samples which contain less than stoichiomet’ric amounts of Mg*+ presumably rapidly bind Mg2+ when added to the assay mix. Catalytic activity was indirectly correlated with Mg*+ content using a partially purified pyrophosphatase preparation which contained active yeast protease. The preparation was treated with 10 rnl\r EDTA in 0.1 &I glycine, pH 9, at 45” C to rapidly inactivate the pyrophosphatase by removal of Mg2+ and proteolysis of the metal-free enzyme. At intervals throughout the inactivation portions were removed and immediately chilled to terminate any further Mg2+ release and proteolysis, and the nIg2+ already released was removed by dialysis. Calculated on the basis of catalytic activity, the initial preparation contained 0.97 Mg2+/mole of enzyme. The results, as shown in Icig. 8, indicate a rough correlation between h1g2+ content and catalytic activity remaining. Thus, in the presence of protease which rapidly inactivates the 1Ig*+-free pyrophos-
0
AND
BUTLER
phatase at high pH and temperature, a partially inactivated sample apparently consists of a mixture of native enzyme containing approximately 1 Mg*+ per mole, and and inactive digestion products which contain little or no nIg*+. Protease-free enzyme was treated with 5 rn>r EDTA at pH 9.0, 30” C, for 24 hr to remove Rig*+, and then the molecular weight (sedimentation equilibrium) was determined after dialysis at 5” C against 0.01 nr Tris, pH 7.4, containing 0.1 JI KCl. A control sample from which EDTA was omitted was fully active, and the molecular weight was 70,000. The EDTA-treated sample was heterogeneous, with an average molecular weight of 39,000 as estimated from the (nonlinear) plot)s of In Z vs 7,2.This is consistent with t’he presence, after extensive treatment with EDTA, of a mixture of 71,000 A/, enzyme dimers and 35,000 M, monomers. These experiments suggest that the strongly bound llIg2+ helps to maintain the enzyme in a dimeric form which is resistant to proteolytic digestion. A kinetic test was devised to more directly cxaminc the correlat’ion between R4g2+ content and catalytic act’ivity. A crystalline pyrophosphat,asc preparation essrntially free
I
I
I
I
I
I
I
I
I
I
5
IO
I5
20
25
30
35
40
45
50
MINUTES
OF
INACTIVATION
AT 45°C
FIG. 8. Correlation of loss of Mg2+ and activity. Partially purified enzyme (sp act 20), 144 mg protein, was incubated at 45°C in 20 ml of 0.1 M glycine, pH 9.0, containing 10 mM EDTA. At intervals 2-ml aliquota were removed, cooled in ice, and placed into dialysis bags which had been boiled in dilute EDTA solution. All samples were dialyzed for 12 hr at 5°C against each of the following buffers successively: 0.1 M Tris, pH 7.3; 0.01 M Tris, pH 7.3, containing 0.1 mM EDTA; 0.01 M Tris, pH 7.3, with Chelex 100 in the dialyzing buffer. After dialysis the samples were assayed for pyrophosphatase activity (dashed lined) by the standard assay and for Mg2+ content (solid line) by atomic absorption.
YEAST
PYROPHOSPHATASE
AND
BOUND
Mg2+
723
of yeast protease activity was incubated under conditions (5 ml1 EDTA, pH 9.0, 30” C) in which Mg2+ is rapidly removed, judging by the susceptibility to proteolysis in ot,hrr preparations on such t’reatment. In hhr normal assay this presumably i\Ig2+dcficicnt enzyme exhibited kinetics linear with timcx, as obscrvcd with native cnzymc (Fig. 9a). However, in assay conditions (0.3 rnlr JIgz+, 2 rnh1 ]‘I’,) where the concentration of free 11g2+ is very low, a definite lag was observed (Fig. 9b), indicating a noninstantaneous recovery of activity. Brief EDTA trcat,ment at pH 7.4 rather than pH 9.0 did not result in a lag in the low-11g2+ assay (Fig. 9c). These observations suggest that pyrophosphatase which does not contain strongly bound RIg2+ is inact,ive but can be rapidly converted to an active form at a rate which is dependent upon the conccntration of free Mg2+. Replacement of Mg2+ in the assay by eit.her lIn2+ or Zn2+ gave low levels of activity as previously observed with these ions as activat,ors (1); no increase in activity was observed with time as was found with _11g2+. It would appear that the only active enzyme in such conditions is that from which JIg2+ had not yet been removed. DISCUSSION
FIG. 9. Effect of assay conditions of Mg2+-deficient enzyme. Protease-free crystalline pyrophosphatase (0.03 mg) obtained by t’he bicarbonate extraction procedure was incubated at 30°C in 0.5 ml of 0.2 M Tris, pH 9.0, containing 5 mM EDTA. At various times small samples were removed and added to 10 ml of assay solution maint,ained at 30°C. One-milliliter portions were removed from this incubation at various times and assayed for orthophosphat,e product,. Figure 9a: standard assay conditions (2 rnM Mg*+, 2 mM PPi); -Obefore enzyme was exposed to EDTA at pH 9; ---O--: 30-min exposure; -a-70-min exposure. Figure 9b: “low Mgz+” assay (0.3 mM Mg2+, 2 mar PPi); -a--, before exposure to EDT.4 at pH 9; ---O-, 15-min exposure; -A-, 45-min exposure. Figure 9c: exposure to 1 rnM EDTA in 0.1 M Tris, pH 7.4 at 30°C. Activity was measured with the “low Mg*+” assajr (0.3 rnM Mg2+, 2 rnM
The original purification of yeast pyrophosphat,ase (1) has been supplanted by a variety of procedures (3, 28, 29); the principal advantage of this procedure is the simple method of crystallization and the stability of the crystals. The molecular weight’ of 71,000 as det’ermined by sediment’ation equilibrium is somewhat greater than the value of 63,000 obtained by Schachman using sedimentation velocity measurements (30). The presence of subunits of molecular weight of approximately 35,000 as found here and in Berlin (31) IS consistent with a 71,000 M, dimcr as the native enzyme. The dimeric sbructure of the molecule correlates well with the previous observations of 2.0 binding sites per 71,000 3, for divalent PPi). illiquots were assayed and 200-min exposure to EDTA; t,he same results as shown.
after 0-, lo-, GO-, all samples gave
RIDLINGTON,
724
YANG,
metal ions4 and for a Ca*+-pyrophosphate complex (4). Avaeva et al. had previously suggested that the enzyme consists of subunits (32). Molecular weights of 56,000, 36,000, and 12,000-17,000 for yeast pyrophosphatase and its subunits were obtained using enzyme chemically modified by maleic anhydride (3’2, 33). It is not known whether these preparations contained protease activity; the relationship of these results to our data is not clear. The fluorescence titrations indicat,e that chelators bind to the enzyme much less strongly than they bind Mg2+ (Table I) ; moreover, the relative strength of binding is different. EDTA binds t’o the enzyme more strongly than does CDTA, which has a somewhat greater affinity for free Mg2+ (10); perhaps this is a consequence of the greater bulkiness of CDTA and geometrical restrictions at the enzyme-binding site. The ext,remely weak binding of pyrophosphatase is in agreement, with direct binding and kinetic studies (4, 5). There appears to be a rough correlation between strength of binding of chelator to the enzyme and degree of enhancement of protein fluorescence (Table I>* We have previously demonstrated two distinct roles for ,1Ig*+ in this reaction: activation of the enzyme by binding free ptIg?+, convert’ing the enzyme to a form which can then bind substrate, which is a llIg2+ complex with pyrophosphate (4, 5). The present’ results suggest a t,hird role for Mg2+, maintenance of the stable native tertiary and quaternary structure of the enzyme. This apparent ‘(structural” Mg2+ is probably not the same as t,he “activating” RIg2+; for the lat.ter there are two binding sites per mole of dimer with K. = 1.7 X 10-j-S X 10P5 ~1 (4), but the structural RIg2+ may have only 1 site per dimer, and dissociation apparently does not occur to an appreciable extent in the absence of chelators. Zn2+, RIn2+, and Co2+ can replace nlg’+ in the first two roles, albeit with resulting lower activit’ies (1) ; kinetic measurements reported here using enzyme treated with EDTA t,o remove Mg2+ suggest that the structural Mg”+ cannot be 4 B. in(28).
Cooperman,
unpublished
results
cited
AND
BUTLER
replaced by Zn*+ or RIn*+ with retention of activit’y. In other experiments (not shown) it was found that incubation of the native enzyme with 1 rnnl Zn2+ or certain rare earths at’ pH 7.2, 30” C, resulted in rapid loss of activity; although Mg2+ and Zn2f analyses were not performed, these observations suggest that the strongly bound Mg2+ can be replaced, but with loss of activity. Nagnesium metalloenzymes are rare but not unknown (34). Scruttin and co-workers recently reported that pyruvate carboxylase from calf liver is a metalloenzyme which contains bound magnesium and bound manganese in a combined stoichiometry equivalent to the biotin content of the enzyme; the same enzyme prepared from livers of chickens raised on a manganese-deficient diet contains bound magnesium rather than bound manganese (35). 11g2+ is better known as a freely dissociable activating ion (36). As early as 1950, however, it was suggested that yea&-type inorganic pyrophosphatases probably contain mangesium (37). No pyrophosphatase has previously been shown to contain strongly bound metal ions, although a role for cobalt (38), manganese (39), or zinc (40) in maint’enance of activity of various bacterial pyrophosphatases has been demonstrated. Particularly interesting with respect to our findings is the latter case in ion while which Mg*+ is the activating Zn”+ is required to maintain the enzyme in an active form (40). Investigation of the yeast enzyme is continuing in our laboratory. ACKNOWLEDGMENTS We are grateful to Dr. B. Cooperman for communicating unpublished results and for suggestions of chromatographic conditions; to Dr. D. Filmer for ultracentrifugal analyses; to Drs. B. Vallee and H. Parker for metal analyses. This work was supported by Research Grants AM 12382 from the National Institllte of Arthritis and Metabolic Diseases and GB 8376 from the Nntional Science Folmdation. LGB is a recipient of a Research Career Development Award (GM 46404) from the U. S. Public Health Service. REFERENCES 1. KUNITZ, 2.
HEPPEL,
Hiol.
(1952). J. Gen. Physiol. 36,423. L. A., AND HILMOE, R. J. (1951) Chem. 192, 87. hf.
J.
YEAST
PYROPHOSPHATASE
RI. (1961) Arch. Biochem. Biophys. 3. KUNITZ, 92,270. L. Cr. J. 4. RIDLINGTON, J. W., AND BUTLER, Biol. Chem. in press. 5. MOE, 0. A., AND BUTLER, L. G. J.Biol.Chem., in press. 0. A., AND BUTLER, L.G. J.Biol.Chem., G. Mel:, in press. 11. E., LI, T. K., AND VALLEIJ, B. L. 7. I)RUM, (1969) Biochemistry 8,3783. Extraction of 8. STARY, J. (1964) in The Solvent lMetal Chelates (Irving, H., ed.), p. 136, Macmillan Company, New York. C. H., AND SUBBARO~, Y. H. (1925) 9. FISKE, J. Biol. Chem. 66,375. G.: AND MARTELL, A. E. (1964) in 10. SILLEN,L. Stability Constants of Met,al-Ion Complexes, p. 697, The Chemical Society, London. Il. JXOBY, W. B. (1968) Anal. Biochem. 26, 295. E. A., GUBLER, C. J., AND KGBY, 12. NOLTMANN, S. A. (1961) J. Biol. Chem. 236, 1225. B. J. (1964) Ann. N. Y. Acad. Sci. 121, 13. DAVIS! 404. A. (1967) J. Biol. 14. TONO, H., AND KORNBERG, Chew 242, 2375. D. L. Submitted to Anal. Biochem. 15. FILMER, (1957) in Methods in HI. K. 16. BCH.4CHMAN, Enzymology (Colowick, S. P , and Kaplan, N. O., eds.), Vo!. 4, p. 70, Academic Press, New York. W. (1952) J. Amer. chef% k’c. 17. HAUSMANN, 74,313s. I., AND OSBORN, M. (1969) J. Biol. 18. WEBER, Chem. 244,4406. J. R. (1970) Biochem. Biophys. Res. 19. PRINGLR, Commun. 39, 46. 20. RUST~M,Y.M.,MASSARO, E. J., ANDBARNARD, E. A. (1971) Biochemistry 10,3509. 21. LOEB, G. I., AND SCHERAGA, H. A. (1956) J. Phys. Chem. 60, 1633. 22. LI, T. K., AND VALLEE, B. L. (1964) Biochemistry 3,869. 23. HARRIS, J. L., AND PERHaM, R. ?;. (1965) J. Mol. Biol. 13,876.
AND
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Mg*’
725
24. EHRENRERG, A. (1957) Acla Chem. Stand. 11, 1257. 25. JOVIN, T., CHRliMBACH, 9., ‘4ND NAUGHTON, RI. A. (1964) Anal. Biochem. 9, 351. 26. BLNESI, H. A., AND HILDILBRAND, J. H. (1949) J. Amer. Chem. Sot. ‘71, 2703. 27. FOSSET, M., MUIR, I,. W., XEILSEN, L. D., AND FISCHER, E. H. (1971) Biochemistry 10, 4105. 28. BUTLRR, L. G. (1971), in The Enzymes (Boyer, P. D., ed.), 3rd edition, Vol. IV, p. 529, Academic Press, New York. 29. NEGI, T., AND IRIB, ill. (1971) J. Biochem. 70, 165. 30. SCHACHMAN, H. K. (1952) J. Gen. Physiol. 36, 451. 31. HOFM~NN, E., AND BOEHME, H.-J. (19il) Fed. Eur. Biochem. Sot. Left. 17, 1. 32. Av.4w.4, S. M., BRAGA, E. A., AND EGOROV, *4. ill. (1968) Biofizika 13,1126. 33. A$v.4cv~, S. M. (1970) Proceeding of the Fifth Anniversary Meeting of the Laboratory of Bioorganic Chemistry, Lomonosov State University, Moscow, December 1970, p. 211. 34. KUBO~~ITZ, F., AND LUTTGENS, W. (1940) Biochena. 2. 307, 170. 35. SCRUTTON, M. C., GRIMINGER, P., AND WALLACB, J. C. (1972) J. Biol. Chem. 247, 3305. 36. VALJ,EL, B. L. (1960) in, The Enzymes (Boyer, P. D., L.IRDP, H. A., .~ND MYRBBCI~, K., eds.), 2nd edition, Vol. 3, p. 225, Academic Press, New York. 37. ROCHE, J. (1950) in The Enzymes (Sumner, J. B., and MYRB&X, K., eds.), 1st edition, Vol. I, Part I, p. 493, Academic Press, New York. 38. OGINSKT, E. L., AND RUMBAUGH, H. L. (1955) J. Bac(erio1. 70,92. 39. TONO, II.? .~ND KORNBERC,, A. (1967) J. Bid. Chenz. 242,2375. 40. KLEMME, J.-H., KLEEME, B., AND GEST, H. (1971) J. Bacleriol. 108, 1122.