Purification and Properties of Phenylalanyl-tRNA Synthetase from Baker" %east1
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AHZALHOSSAIN" Depnrtnient of Biology, University of Perarzsylvania, Philadelphia, Pennsylr~aniad 91 74
Received May 266, 1975 Hossain, A. (1955) Purification and Properties of Phenylalanyl-tRNA Synthetase from Baker's Yeast. Can. 9. Biocllem. 53, 1316-1322 In an effort to avoid proteolytic fragmentation of enzymes extracted from yeast cells, the (L-phenylalanine:tRNAPhe ligase (AMP-forming) phenylalanyl-tRNA synthetase (EC 6.1.1.20) ) has been isolated from toluene lysates of baker's yeast in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride. The procedure includes ammonium saalfate fractionation and chromatography on DEAE-cellulose and hydroxylapatite columns. AcryEamids gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate indicates a single subunit of 75 000; other isolations have led to two subunits of 75 008 and 63 080, respectively, in agreement with other workers. Steady state kinetic analysis of the enzyme has also been carried out. The apparent kinetic patterns resulting from application of Cleland9sprocedure, in which the substrates are varied pairwise in the presence of satlarating concentrations of the third component, suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion but do not completeEy eliminate a random mechanism. Hossain, A. (1975) Purification and Properties of Phenylalanyl-tRNA Syanthetase from Baker's Yeast. Carz. 9. Bioci'lem. 53. 1316-1 322 Dans Ee but d'Bviter la fragmentation prottolytique des enzymes extraites des cellules de la levure, nous avons is016 la phCnylalanyl-tRNA synthktase (EC 6.1-1.20) des lysats toluCniques de la levure de boulangesie en prksence d'un ianhibiteur de la prothase, le phtnylm6thylsulfonyl Auorure. La technique comprend aane fractionnation par le sulfate d'ammonium et la chromatographie sur colonnes de DEAE-cellulose et d'hydroxylapatite. L9Clectrophorbsesus gel d'acrylamide de I'enzyme, en prCsence de dodCcyl sulfate de sodium, rCvele une seule sous-unit6 de 75 800; d'accord avec d'aaatres chercheurs, des techniques diffCrentes d9isolation nous ont conduit h deux sous-mitts, l'une de 75 0660 et I'autre de 63 000, Nous avons poursuivi l'analyse cinCtique de %'enzymea 1'Ctat stationnaire. Les profils cinktiques apparents obtenus par application de la technique de Cleland 021 les substrats varient deux par deux en prksence de concentrations saturantss d'un troisikme compos6, suggerent un m6canisme de rkaction dans lequel I'ATP et la phCnylalanine entrent dans la rkaction seIon un ordre obligatoire mais n'eliminent pas complbtement le mCcanisme de h a a r d . [Traduit par le journal]
Introdnetion The phenylalanyl specific transfer RNA from yeast (tRNAphe) has been subjected to more intensive investigation than any similar tRNA molecule, due to its availability and favorable crystallization properties and possession of a naturally fluorescent base in the anticodon loop. Its primary structure has been completely determined ( 1, 2 ) , and its conformation in a crystal has been reported (4, 5 ) . An extensive series of chemical and functional studies in so'This investigation was supported by NSF grant GB-292 10. 'Present address: Department of Biochemistry, McGill University, Montrealal,Quebec H3G 1Y6.
lution has been performed, including analysis sf fluorescence transfer in the molecule ( 6 ) ,circular dichroic measureinents (7), chemical modification experiments ( 8 ) , complementary oligonucleotide binding (9), and enzymatic or chemical fragmentation and reconstitution ( 10). It is natural that considerable effort has been devoted to isolating and characterizing the cognate aminoacyl synthetase, yeast phenylalanyl-tRNA synthetase" (PRS) . Several reports describing the preparation and properties of this enzyme have appeared ( 11-1 3 ) ; two sf these ( 11, 12) concur in identifying a azPa3~-PIaenylalanine: tRNAPhe ligase (AMP-forming ) (EC 6.1.120)a
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HOSSAIN: YEAST PHENYLALANYL-tRNA SYNTHETASE
tetrameric subunit structure for PRS. However, as described more fully below, these give substantively different accounts of the size of the subunits, as well as of the specific activity and stoichiometry of substrate binding. We describe here the isolation of PRS from baker's yeast using toluene lysis which yields an enzyme of high specific activity. We interpret the differences among various isolates of PRS as reflecting a degree of proteolytic cleavage in this species as has already been found in the case of yeast hexokinase (EC 2.7.1.1 ) (14), for example. Partial purification of yeast PRS was first reported by Eagerkvist and Waldenstrom ( 15) ; subsequently Makman and Cantoni (16) obtained a 180-fold purification of the enzyme as a by-product of a procedure for preparing yeast seryl-tRNA synthetase (EC 6.1.1.1 1 ) .The estimated molecular weight of this material was 1.8 X 1OValtons. Eater preparations, with molecular weights estimated at 2.2 X lo5 (1 1) and up to %.$ X 18" 12), respectively, had higher specific activities, although some discrepancies in the she of the subunits have been noted. Fasiolo et al. (11) report subunits of 5.6 X 1 0 h n d 6.3 X lo4 daltons while the preparation of Reid et al. (12) yielded 6.3 X 1 0 h n d '7.5 X lo4. Since the former workers used laboratory cultured yeast, and the latter a commercial source, it is possible that these differences arise from differences in the strains. hkaeeharomyees eerevisiae is not genetically homogeneous. Utilizing a commercial source similar to that of Reid et al. ( I % ) , we find that the product occasionally yields a polyacrylarnide gel electrophoretic pattern indicating a single subunit of molecular weight 7.5 x 10% similar to that of the larger subunit of these workers ( 11, 12). A second subunit of 65 000 daltons is detected at other times, similar to the report of other workers (12). The starting point for the procedure described here was the observation that toluene lysis does not inactivate certain tRNA synthetases from baker's yeast, including the histidyltRNA synthetase (EC! 6.1.1.2 1) ( 17), lysyltRNA synthetase (EC 6.1.1.6) ( I $ ) , and the tryptophanyl species. Assay of a toluene lysate obtained in a preparation of the tryptophanyltRNA synthetase (EC 6.1.1.2) (19) showed that substantial phenylalanyl activity was present.
Remaining stages of the procedure are patterned after the methods of Reid et al. ( 12), and Tener and von Tigerstrom ( 17), except for inclusion of a proteolytic enzyme inhibitor in most steps, since these lysates retain some prsteolytic capacity.
Materials and Methods Materials Baker's yeast was purchased locally. Unfractionated brewer's yeast tRNA and tRNAPhe were purchased from Boehringer-Mannheim, DEAE-cellulose (capacity 0.U mequiv.ig) was obtained from Sigma Chemicals. Hydroxylapatite was purchased from Bio-Rad Laboratories (Bio-Gel HTP, control No. 6709). L-[14@]PhenylaIanine (455 mCiimmol, 99% radioactivity) was a product of Schwarz BisResearch. Phenylmethylsulfonyl fluoride was obtained from Calbiochem Inc. Protein markers were commercially obtained. All other chemicals were reagent grade compounds.
>
A S S O ~Procedures In column eluates protein was determined by absorbance at 280 nm. Protein concentrations in the pooled fractions were determined by the method of Lowry et al. (20) with bovine serum albumin as standard. Aliquots of the crude extract in the early stages of purification were dialyzed before analysis to remove low molecular weight compounds which interfere with the Lowry assay. Proteslytic Enzyme Proteolytic enzyme activity in the extracts at each stage of purification was determined on a casein substrate by the method of Laskowski (21). Broteolysis was also determined by incubating aliquots sf enzymic extracts with casein, precipitating the undegraded casein with perchlorate and meastaring the yellow color remaining in solution in alkaline medium at 428 nm. Aminocacybcation Enzymic activity was assayed at the pH optimaam for aminoacylation in a reaction mixture which contained (in 0.2 ml) 20 pmol of Tris-HCl (pH 8.0, titrated at 25 "C), 10 pmoH of MgC12, 2 pmol of ATP, 2 pmol of reduced glutathione, 2 mg of yeast tRNA. 20 pg of tRNAPhe, 0.025 pmol of L-['*C]phenylalanine (455 mCi/mmol) and an appropriate aliquot of enzyme. The reaction mixture was incubated at 37 "C and 50 p1 were withdrawn after 5-45 min, pipetted onto filter paper discs (Schleicher and Schuell No. 593-A) which were dropped immediately into ice-cold trichloraceeic acid and washed and counted according to the procedure of Schmidt and Reid (22). The optimum conditions were determined experimentally. Gel Electrophoresis Enzyme preparations were dialyzed into 5 rn~Vpotassium phosphate (pH 7.2), 2.5 mM mercaptoethanol. 0.5 mM EDTA, and 10% glycerol and then dissociated into subunits by incubating in 10 mM sodium phos-
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CAN. J. BIOCWEM. VOL. 53, 1975
phage (pH 7.0), 1% sodium dodecyl sulfate (SDS), and 1% mercaptoethanol, according to Weber and Osborn (23). After incubation, the protein solutions were dialyzed for several hours at room temperature against 500 ml of 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1 % SDS and 0.1% 2-mercaptoethanol. Electrophoresis was carried out essentially as described in the above references with the following minor changes. Gels were polymerized with one-half the normal amount of cross-linker, gels of 15 ml volume were poured into tubes of 6 mm inside diameter. Separation was performed at 8 mA per gel for 4 h using bovine serum albumin, ovalbumin, DWAse, and 8-galactosidase (EC 3.2.1.23). Enzyme Purification All operations were carried out at 4 "C, unless otherwise noted. Step 1: Extraction The procedure of von Tigerstrom and Tener (14) was followed with some modifications. Toluene (200 ml) was placed in a 1 litre Erlenmeyer flask and heated in a water bath to 45 "G. Then 1 lb (8 lb = 0.45 kg) of fresh baker's yeast and 0.60 g cysteine hydrochloride dissolved in 825 ml of 0.65 M Tris-acetate buffer (pH 8.0) containing the protease inhibitor, phenylmetbylsulfonyl fluoride (PMSF) at a concentration of 508 pg/100 ml solution were added. The contents of the flask were stirred occasionally until the temperature was rose to approximately 37 "C whereupon @02 evolved and the yeast started to liquefy. The mixture was kept at 37 "C for 2 h with occasjonal stirring, cooled to 4 "C, and 180 ml of cold 0.01 M Trisacetate, pH 8.5, were added. After 3 h at 4 "C, the extract was siphoned from below the emulsion and the pH maintained at 7.5 by addition of 1 M Trisacetate buffer, pH 8.5. Protease inhibitor was added and the suspension centrifuged at 10 000 rpm in a Sorvall RC 2-B centrifuge for an hour at 4 "C. The resulting supernatant (200 ml) was removed. A small aliquot was removed for dialysis and activity assay, and the remainder immediately subjected to ammonium sulfate fractionation. Step 2: Arnmonium Sulfate FracEionabion Solid ammonium sulfate was added with stirring over a period of 30rnin to 50% saturation at 4 "C. Stirring was continued for an additional 30 min before centrifugation (10 000 rpm for 1 h). The supernatant was adjusted to 70% saturation by the gradual addition of solid ammonium sulfate. After stirring for another 30 min, the suspension was centrifuged at 10 000 rpm for B h. The precipitate obtained was dissolved in a small volume ( 18 ml) of 10 mM potassium phosphate (pH 7.2)' 5 mikf mercaptoethanol, 1 mild EDTA, and 10% propylene glycol, then PMSF was added and the solution was dialyzed with three changes of the same buffer to remove excess salt. Step 3: DEAE-cellulose Chromatography Preparation of Column DEAE-cellulose was suspended in 2.0 M ammonium carbonate poured into a column with a diameter of 2.5 cm, and packed to a height of 30 cm. It was washed
10
20 FRACTIONS
30
FIG. 1, Chromatographic purification of ammonium sulfate fraction on a column of DEAE-cellulose in the presence of 35:& (v/v) propylene-glycol as described in Materials and Methods. Azsoand radioactivity are represented by closed and open circles, respectively. with 2 M ammonium carbonate until the optical density of the eluates at 260 nm was zero, then with several bed volumes of 0.1 M ammonium carbonate containing 0.1 M EDTA and finally with water until the eluate was neutral. The cellulose was converted to the phosphate form with 0.5 M potassium phosphate (pH 7.2) and then washed with 0.08 M potassium phosphate buffer (pH 7.2) containing 5 mM mercaptoethanol, 1 mM EDTA, 35% propylene gIycol, and protease inhibitor (500 pg/100 ml solution). The dialyzed product from step 2 was then applied and the column washed with 50 an1 of the above buffer. Elution was carried out with a linear gradient from 80 to 320 mM potassium phosphate (pH 7.2), both buffers containing 5 mM mercaptoethanol, 1 mM EDTA, 35% propylene glycol. and PMSF inhibitor. Flow rates were maintained at approximately 1 ml/min. Fractions of 20 ml were collected. Enzymic activity was eluted at a phosphate concentration of 0-1 M (Fig. 1). Fractions containing PRS3 activity were pooled (I00 ml) . Step 4: Hydroxylapatite Chronzatography The pooled fractions were applied to a 3.2 cm x I0 cm column of hydroxylapatite and powdered cellulose (4: I, w/w ) equilibrated with 10 mM potassium phosphate (pH 7.2), 5 mM mercaptoethanol, 1 mM EDTA, 20% propylene glycol, and PMSF. After application of the sample, the column was eluted with a linear gradient of 180-300 ml potassium phosphate (pH 7.21, both buffers containing 5 mM mercaptoethanol, l mM EDTA, 20% propylene glycol, and PMSF. The bulk of the protein elutes early while the enzj7meappears approximately half way in the gradient (Fig. 2). The active fractions (50 ml) were pooled, diluted with 2 volumes of H20, stirred gently and applied to a hydroxylapatite column (2 m x 3 cm); elution was carried out with a minimum volume of 0.5 Ad potassium phosphate (pH 7.2), 5 mM EDTA, and 10% propylene glycol. This step was suggested by Dr. B. Reid, as giving greater yields than over the final ammonium sulfate step of Reid et al. (12). The purified enzyme was either used for analysis or stored at
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HOSSAIN: YEAST
PHENYLALANYL-tRNA SYNTHETASE
FRACTIONS
FIG.2. Gradient elution of BEAE-cellulose fraction on a column of hydroxylapatite by the procedure outlined in Materials and Methods. A280 and radioactivity are represented by closed and open circles, respectively. TABLE1. Purification of Phe-tRNA synthetase (PRS) Fraction 1. 2. 3. 4.
Post-mitochondria1supernatant (Nfi)aSOr fraction DEAE-cellulose fraction Hydrsxylapatite fraction (final enzyme)
Protein (mg)
Specific activity (cpm/rng protein)
1480 674 12.4 0.5
0.003 0.005 0.12 2.1
X 10' X 10V X106 X 106
Total activity 4.8 X 10" . 3 7 X lo6 1.488X106 1.05 X 106
Recovery Purification
(5%)
1 1.67 40
100 69.9 30.9 21.7
700
TABLE 2. Proteolytic contamination as assayed by the casein digestion method at various stages sf purification Sample 1. 2. 3. 4.
Post-mitochondria1supernatant (NIf4)2S04fraction BEAE-cellulose fraction Hydroxylapatite fraction
~ ~ ~ of ~ protein / m g
Azsr/mg of protein
Ar2a/mg of protein
0.243 0.203 0.008 0 .MI8
Proteolytic Activity of the Enzyme Fractions Proteolytic enzyme activity in the extracts at each stage of purification was determined on the Kinetic Measurements Activity of PRS was assayed by measuring the casein substrate by the methods outlined in incorporation of L-rPC]phenylalanine into tRNAPfae 'Materials and Methods'. The results in Table 2 as described earlier in this text. Initial velocities were obtained using concentrations which ensure a linear indicate that this extraction procedure leads to relationship between reaction rates and incubation greatly diminished (although measurable) time. All kinetic constants were fitted to data by a levels of proteolytic activity in the early phases; numerical procedure of Dr. Richard Viale, using a in later stages no proteolytic activity can be dePDP-10 computer. The rate patterns were deduced tected on the casein substrate used. from inspection of Lineweaver-Burk reciprocal plots constructed from the numerically derived constants. Assessment o f Purity and Determination o f Typical experiments were run for severd fixed conMolecular Weight centration values of a second reactant in the presence The purified enzyme obtained after hydroxsf a saturating concentration of a third reactant. ylapatite chromatography was subjected to analysis by gel filtration and electrophoresis. Results Figure 3 shows the elution profile obtained by Puri fic~tion chromatography on a column of Bio-Gel A The phenylalanine-tRNA synthetase (PRS ) (1-5 m ) using buffers containing 0.5 M ammohas been purified 700-fold. nium sulfate. The preparation appears to be
-20 after mixing with an equal volume of propylene glycol. OC.
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CAN. J .
BHOCHEM. VOL. 53.
1975
FRACTIONS
FIG.3. Gel filtration of purified PRS. Purified enzyme (2.5 mg)in 3 ml of 10 m M potassium phosphate (pH 7.2), 5 mM mercaptoethanol, 1 mM EDTA, and 1O& : glycerol was applied to a column (I X 78 cm) of Bio-Gel A (8.5 m) equilibrated in the same buffer containing 0.5 M ammonium sulfate. Fractions of 1.5 ml were collected, monitored for absorbance at 280 nm, and assayed for PRS activity as described in the text. The arrows refer to the elution positions of (I) blue dextran (BD) (rnol. wt. 2 X 80"; (2) rabbit muscle pyruvate kinase (PM); (3) lactic dehydrogenase (LDM) (msl. wt. 150 088); and (4) ovalbumin (QA) (mol. wt. 43 OW).
FIG.5. Electrophoresis of purified PRS in polyacrylamide gels containing SDS and estimation of the mol. wt. of its subunits. The enzyme was dissociated into subunits and subjected to the conditions described in Materials and Methods; 10pg of enzyme used. The molecular weight of the protein markers are plotted, on a logarithmic scale, against their mobilities on acrylamide gels during electrophoresis in the presence of SDS. The proteins are pgalactosidase (@-gal,130 00); serum albumin (SA, 68 W); ovalbumin (CIA, 43 000); and DNA* (31 000).
data according to the method of Andrew (24) as shown in Fig. 4 yields a value sf approximately 350 000. Sodium Dodecyl Sulfate (SDS) Gel EBectrophoretic Studies of PRS Purified PRS was dissociated into subunits in SDS-mercaptoethad and subjected t s elecFIG.4. Estimation of the molecular weight of PWS in trophoresis in pslyacrylamide gels containing Bio-Gel A (1.5 m) as described in the text. The molecular SDS using the method of Weber a d Bsborne weight of the protein markers are plotted, on a logarithmic ( 2 3 ) with the modifications described in 'Mascale, against their K a ,, the par tition coefficient between terials and Methods'. As in Fig. 5 , a single band the liquid phase and the gel phase. K,, = (V, - Vo)j was observed under standard electrophoretic (V, - Vo), in which V, is the elution volume of the sample, V, the total volume of the gel bed, and Vo the conditions. Calculation of the molecular weight void volume evaluated by blue dextran. Pyruvate kinase of this band by comparison to the relative mo(PM, mol. wt. 230 000 - 237 000); lactic dehydrogenase bilities yields a value close to 75 000. A second (LDH, rnol. wt. 150 OW); and ovalbumin (QA, mol. wt. subunit of 65 800 daltons has also been de43 m). tected sometimes similar to that observed by Reid et al. ( I 2 1. quite homogeneous as evidenced by a single protein peak of constant specific activity. Also Initial Velocity Studies shown in Fig. 3 are the elution positions of An intersecting rate pattern was observed known msIecular weight standard proteins after when %/v was plotted against 1/[Phe] for exchromatography on the same column. Estima- periments done at different fixed Bevels of ATP tion of the molecular weight of PRS from these (Fig. 6A). A family sf lines intersecting to the
HOSSAIN: YEAST PMBNYLALANYL-tRNA SYNTHETASE
Al - fixed ATP
l e ~ l s
I
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ATP
- Varied
i3
-
~ R N A fixed ~ ~ levels ATP
-
saturated levels
8 Phe
- SATURATED LEVELS
"0, 10
.,
FIG.6. Initial velocity patterns (A) with phenylalanine as the variable substrate at different fixed concentrations of 1.5 m M ATP); (B) with ATP as thevariable substrate at ATP at saturating levels of tRNAPhe ( e , 0-75 m M ATP; 0, 50 pM Phe; different fixed concentrations of phenylalanine at saturating levels of tRNAPhe ( e , 25 yM Phe; 8, 100 pM Phe); ( 6 )with phenyialanine as the variable substrate at different fixed concentrations of tRNA at saturating concentrations sf ATP ( e , 0.2 pM tRNAPhe; 8 , 0.5 pcMtRNAPhe; A, 1.8 pM tRNAPhe); (19) with ATP as the variable substrate at different fixed concentrations sf tRNA at saturating levels of phenylalanine ( e , 0.25 pM tRNA; 8 , 0.5 pM tRNA; 18 pM tRNA).
.,
left of the vertical axis was noted when l / v was plotted against B/[ATP] for experiments done with different concentrations of L-phenylalanine and with saturating levels of tRNAghe (Fig. 6B). From an inspection of Fig. 6 A and B, it can be seen that both the slope and the intercepts vary. This suggests that the variable and fixed substrate may combine with different enzyme forms, which are reversibly connected. According to Cleland (261, this pattern suggests a mechanism in which substrate ATP and phenylalanine binds to the enzyme before any product can be formed.
When phenylalanine was the variable substrate at several fixed concentrations of tWNAPb and saturating ATP concentrations, the double reciprocal plots exhibit a parallel pattern (Fig. 66.). This pattern also resulted when ATP was varied at different fixed concentrations of tRNAPhe (Fig. 6D). Non-intersecting patterns are consistent with a Ping-Pong mechanism in which both variable and changing fixed substrate combine with different enzyme forms not interconnected through reversible steps. The rouglaly linear patterns which were evaluated by an inspection of the slope and
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CAN. J. BIOCHEM.
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intercepts of Fig. 6 A and B probably suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion, but do not, however, completely eliminate a random mechanism. Discussion The toluene lysis procedure described above provides a means of isolating yeast P R S q n a highly active form. In essence, our procedure represents a hybrid between that applied by von Tigerstrom and Tener ( 17) to the yeast histidyl-tRNA synthetase (EC 6.1.1.21 ) (partially purified) and the very successful preparation method for PRS due to Reid et al. ( 12). In particular, we have availed ourselves of a modification of the last step in this latter procedure due to Dr. Reid. The most obvious explanation which accounts in a general way for the variability among the published accounts of these enzymes is the presence of proteolytic activity in the yeast. Commercial preparations of yeast hexokinase (EC 2.7.1.1 ) , for example, have been found to be contaminated with traces of proteolytic activity which has complicated determination of the size of the polypeptide subunit of this enzyme (25). Assay of proteolysis using casein indicates that even in the presence of phenylmethylsulfonyl fluoride and following toluene lysis some activity remains (see Table 2). Nevertheless, the present procedure offers a route to reliable preparation of the enzyme on a large scale, as would be essential for the preparation of crystals or detailed investigation of the interaction between the enzyme and the tRNAP". For a three substrate, three product reaction (ter-ter mechanism) there exist six possible mechanisms which may be either sequential or Ping-Pong. Aminoacyl-tRNA synthetases reported so far demonstrate both kinetic patterns (25-27). While the results of this investigation with PRS are superficially consistent with an ordered binding process with ATP first, the actual mechanism cannot be established unequivocally by steady-state kinetic experiments and may be considerably more complex. The investigation by fluorescence equilibrium measurements of the interactions of PRS with its ligands will be reported in detail elsewhere.
VOL. 53.
1975
1. Raj Bhandary, U. L. & Chang, S. H. (1968) J. Biol. Chem. 243, 598-668 2. Philipsen, P., Thiebe, R., Wintermeyer, W.& Zachau, H. G . (1968) Biocherem. Biophys. Res. Commm. 33, 922-926 3. Suddath, F. L., Quigley, G. L., McPherson, A., Sneden, D., Kim, J. J., Kim, S. H. & Rich, A. (1974) Nature (Eondon) 248, 20-24 4. Kim, S. H., Sussman, J. L., Suddath, F. L., Quigley, G. J., McPherson, A., Wang, A. H. J., &man, N. 6. & Rich, A. (1974) Proc. Natl. Acud. Sci. U.S.A. 71, 4970-4974 5 . Rskrtus, J. D., h d n e r , J. E., Finch, J. F., Rhoda, D., Brown, R. D., Clark, B. F. C. & Klug, A. (1974) Nature (Latadon) 250, 54.6-551 6. Romer, R., Reisner, D. & Maass, G. (1970) FEBS b t t . 10, 352-357 7. Blum, A. D., Uhlenbeck, 8.D. & Tinms, I., Jr. (1972) Biochemistry 11, 324-3256 8. Cramer, F., Dmpner, H., vonder Waar, F., Schlimme, E. & Seidel, H. (1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1384-1391 9. Pongs, O., Bald, R. & Reinwald, E. (1973) Eur. J. Bioehem. 32, 117-125 10. Thiebe, R., H a r k n , K. & Zachau, H. G. (1972) Eur. J. Bioehem. 26,132-143 11. Fasiolo, F., Befort, N.,Boulanger, Y. & E k l , J. P. (1978) Biochim. Biophys. Actu 21 7, 305-3 18 12. Schmidt, J., Wang, R., Stanfield, S. & Reid, B. R. (197 1) Biochemistry 10, 3264-3268 13. Rue, B., Surover, M. & Dudock, D. (1973) Biochemistry 12, 41464154 14. Lazarus, N. R., Ramel, A. W., Rustum, Y. M. & Barnard, E. A. (1966) Biochemistry 5, 4003-4025 15. Lagerkvist, U. & Waldenstrom, J. (1964) J. Biol. 8, 28-37 16. Makrnan, M. H. & Cantoni, G . L. (1965) Biochemktry 4,1434-1442 17. von Tigerstrom, M. & Tener, G . M. (1967) C m . J . Biochem. 45, 1067-1074 18. Chulmecka, V., von Tigerstrum, M., Obrenan, P. D. gL Smith, C. J, (1969) J. B i d . Chem. 244, 5481-5488 19. Hossain, A. & Kallenbach, N.R. (1974) FEBS Lett. 45, 282-285 20. Lowry, 0. H., R~senbrough,N. J., Faar, A. L. Br Randal, R. J. (1951) J. Biol. Chem. 193, 265-271 21. hskowsky, M. (1956) Metho& E~mzymol.2, 33-34 22. Schmidt, J. & Reid, B. R. (1971) Anal. Bioehem. 39, 162-1 69 23. Weber, K. & Osborn, M. (1969) J. Biol. Cltem. 244, 44064412 24. Andrews, B. (1965) Bioehcm. J. 96, 595-6435 25. Pringle, J. R. (1978) Biochem. Biophys. Res. Commun.
39,4650 26. Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-1 37 27. Penneys, N. S. & K. H. Muench (1974) Biochemistry 13, 54Q-571 28. Papas, T. S. & Peterkofsky, A. (1972) Biochemistry 11, 4602-4608 29. Papas, T. S. & Mehler, A. H. (1971) J. Biol. CItem. 246,5924-5928 30. Santi, D. V., Danenberg, P. V. & Satterly, P. (1971) Biochemistry 18, 4804-4820
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AHZALHOSSAIN" Depnrtnient of Biology, University of Perarzsylvania, Philadelphia, Pennsylr~aniad 91 74
Received May 266, 1975 Hossain, A. (1955) Purification and Properties of Phenylalanyl-tRNA Synthetase from Baker's Yeast. Can. 9. Biocllem. 53, 1316-1322 In an effort to avoid proteolytic fragmentation of enzymes extracted from yeast cells, the (L-phenylalanine:tRNAPhe ligase (AMP-forming) phenylalanyl-tRNA synthetase (EC 6.1.1.20) ) has been isolated from toluene lysates of baker's yeast in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride. The procedure includes ammonium saalfate fractionation and chromatography on DEAE-cellulose and hydroxylapatite columns. AcryEamids gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate indicates a single subunit of 75 000; other isolations have led to two subunits of 75 008 and 63 080, respectively, in agreement with other workers. Steady state kinetic analysis of the enzyme has also been carried out. The apparent kinetic patterns resulting from application of Cleland9sprocedure, in which the substrates are varied pairwise in the presence of satlarating concentrations of the third component, suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion but do not completeEy eliminate a random mechanism. Hossain, A. (1975) Purification and Properties of Phenylalanyl-tRNA Syanthetase from Baker's Yeast. Carz. 9. Bioci'lem. 53. 1316-1 322 Dans Ee but d'Bviter la fragmentation prottolytique des enzymes extraites des cellules de la levure, nous avons is016 la phCnylalanyl-tRNA synthktase (EC 6.1-1.20) des lysats toluCniques de la levure de boulangesie en prksence d'un ianhibiteur de la prothase, le phtnylm6thylsulfonyl Auorure. La technique comprend aane fractionnation par le sulfate d'ammonium et la chromatographie sur colonnes de DEAE-cellulose et d'hydroxylapatite. L9Clectrophorbsesus gel d'acrylamide de I'enzyme, en prCsence de dodCcyl sulfate de sodium, rCvele une seule sous-unit6 de 75 800; d'accord avec d'aaatres chercheurs, des techniques diffCrentes d9isolation nous ont conduit h deux sous-mitts, l'une de 75 0660 et I'autre de 63 000, Nous avons poursuivi l'analyse cinCtique de %'enzymea 1'Ctat stationnaire. Les profils cinktiques apparents obtenus par application de la technique de Cleland 021 les substrats varient deux par deux en prksence de concentrations saturantss d'un troisikme compos6, suggerent un m6canisme de rkaction dans lequel I'ATP et la phCnylalanine entrent dans la rkaction seIon un ordre obligatoire mais n'eliminent pas complbtement le mCcanisme de h a a r d . [Traduit par le journal]
Introdnetion The phenylalanyl specific transfer RNA from yeast (tRNAphe) has been subjected to more intensive investigation than any similar tRNA molecule, due to its availability and favorable crystallization properties and possession of a naturally fluorescent base in the anticodon loop. Its primary structure has been completely determined ( 1, 2 ) , and its conformation in a crystal has been reported (4, 5 ) . An extensive series of chemical and functional studies in so'This investigation was supported by NSF grant GB-292 10. 'Present address: Department of Biochemistry, McGill University, Montrealal,Quebec H3G 1Y6.
lution has been performed, including analysis sf fluorescence transfer in the molecule ( 6 ) ,circular dichroic measureinents (7), chemical modification experiments ( 8 ) , complementary oligonucleotide binding (9), and enzymatic or chemical fragmentation and reconstitution ( 10). It is natural that considerable effort has been devoted to isolating and characterizing the cognate aminoacyl synthetase, yeast phenylalanyl-tRNA synthetase" (PRS) . Several reports describing the preparation and properties of this enzyme have appeared ( 11-1 3 ) ; two sf these ( 11, 12) concur in identifying a azPa3~-PIaenylalanine: tRNAPhe ligase (AMP-forming ) (EC 6.1.120)a
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HOSSAIN: YEAST PHENYLALANYL-tRNA SYNTHETASE
tetrameric subunit structure for PRS. However, as described more fully below, these give substantively different accounts of the size of the subunits, as well as of the specific activity and stoichiometry of substrate binding. We describe here the isolation of PRS from baker's yeast using toluene lysis which yields an enzyme of high specific activity. We interpret the differences among various isolates of PRS as reflecting a degree of proteolytic cleavage in this species as has already been found in the case of yeast hexokinase (EC 2.7.1.1 ) (14), for example. Partial purification of yeast PRS was first reported by Eagerkvist and Waldenstrom ( 15) ; subsequently Makman and Cantoni (16) obtained a 180-fold purification of the enzyme as a by-product of a procedure for preparing yeast seryl-tRNA synthetase (EC 6.1.1.1 1 ) .The estimated molecular weight of this material was 1.8 X 1OValtons. Eater preparations, with molecular weights estimated at 2.2 X lo5 (1 1) and up to %.$ X 18" 12), respectively, had higher specific activities, although some discrepancies in the she of the subunits have been noted. Fasiolo et al. (11) report subunits of 5.6 X 1 0 h n d 6.3 X lo4 daltons while the preparation of Reid et al. (12) yielded 6.3 X 1 0 h n d '7.5 X lo4. Since the former workers used laboratory cultured yeast, and the latter a commercial source, it is possible that these differences arise from differences in the strains. hkaeeharomyees eerevisiae is not genetically homogeneous. Utilizing a commercial source similar to that of Reid et al. ( I % ) , we find that the product occasionally yields a polyacrylarnide gel electrophoretic pattern indicating a single subunit of molecular weight 7.5 x 10% similar to that of the larger subunit of these workers ( 11, 12). A second subunit of 65 000 daltons is detected at other times, similar to the report of other workers (12). The starting point for the procedure described here was the observation that toluene lysis does not inactivate certain tRNA synthetases from baker's yeast, including the histidyltRNA synthetase (EC! 6.1.1.2 1) ( 17), lysyltRNA synthetase (EC 6.1.1.6) ( I $ ) , and the tryptophanyl species. Assay of a toluene lysate obtained in a preparation of the tryptophanyltRNA synthetase (EC 6.1.1.2) (19) showed that substantial phenylalanyl activity was present.
Remaining stages of the procedure are patterned after the methods of Reid et al. ( 12), and Tener and von Tigerstrom ( 17), except for inclusion of a proteolytic enzyme inhibitor in most steps, since these lysates retain some prsteolytic capacity.
Materials and Methods Materials Baker's yeast was purchased locally. Unfractionated brewer's yeast tRNA and tRNAPhe were purchased from Boehringer-Mannheim, DEAE-cellulose (capacity 0.U mequiv.ig) was obtained from Sigma Chemicals. Hydroxylapatite was purchased from Bio-Rad Laboratories (Bio-Gel HTP, control No. 6709). L-[14@]PhenylaIanine (455 mCiimmol, 99% radioactivity) was a product of Schwarz BisResearch. Phenylmethylsulfonyl fluoride was obtained from Calbiochem Inc. Protein markers were commercially obtained. All other chemicals were reagent grade compounds.
>
A S S O ~Procedures In column eluates protein was determined by absorbance at 280 nm. Protein concentrations in the pooled fractions were determined by the method of Lowry et al. (20) with bovine serum albumin as standard. Aliquots of the crude extract in the early stages of purification were dialyzed before analysis to remove low molecular weight compounds which interfere with the Lowry assay. Proteslytic Enzyme Proteolytic enzyme activity in the extracts at each stage of purification was determined on a casein substrate by the method of Laskowski (21). Broteolysis was also determined by incubating aliquots sf enzymic extracts with casein, precipitating the undegraded casein with perchlorate and meastaring the yellow color remaining in solution in alkaline medium at 428 nm. Aminocacybcation Enzymic activity was assayed at the pH optimaam for aminoacylation in a reaction mixture which contained (in 0.2 ml) 20 pmol of Tris-HCl (pH 8.0, titrated at 25 "C), 10 pmoH of MgC12, 2 pmol of ATP, 2 pmol of reduced glutathione, 2 mg of yeast tRNA. 20 pg of tRNAPhe, 0.025 pmol of L-['*C]phenylalanine (455 mCi/mmol) and an appropriate aliquot of enzyme. The reaction mixture was incubated at 37 "C and 50 p1 were withdrawn after 5-45 min, pipetted onto filter paper discs (Schleicher and Schuell No. 593-A) which were dropped immediately into ice-cold trichloraceeic acid and washed and counted according to the procedure of Schmidt and Reid (22). The optimum conditions were determined experimentally. Gel Electrophoresis Enzyme preparations were dialyzed into 5 rn~Vpotassium phosphate (pH 7.2), 2.5 mM mercaptoethanol. 0.5 mM EDTA, and 10% glycerol and then dissociated into subunits by incubating in 10 mM sodium phos-
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CAN. J. BIOCWEM. VOL. 53, 1975
phage (pH 7.0), 1% sodium dodecyl sulfate (SDS), and 1% mercaptoethanol, according to Weber and Osborn (23). After incubation, the protein solutions were dialyzed for several hours at room temperature against 500 ml of 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1 % SDS and 0.1% 2-mercaptoethanol. Electrophoresis was carried out essentially as described in the above references with the following minor changes. Gels were polymerized with one-half the normal amount of cross-linker, gels of 15 ml volume were poured into tubes of 6 mm inside diameter. Separation was performed at 8 mA per gel for 4 h using bovine serum albumin, ovalbumin, DWAse, and 8-galactosidase (EC 3.2.1.23). Enzyme Purification All operations were carried out at 4 "C, unless otherwise noted. Step 1: Extraction The procedure of von Tigerstrom and Tener (14) was followed with some modifications. Toluene (200 ml) was placed in a 1 litre Erlenmeyer flask and heated in a water bath to 45 "G. Then 1 lb (8 lb = 0.45 kg) of fresh baker's yeast and 0.60 g cysteine hydrochloride dissolved in 825 ml of 0.65 M Tris-acetate buffer (pH 8.0) containing the protease inhibitor, phenylmetbylsulfonyl fluoride (PMSF) at a concentration of 508 pg/100 ml solution were added. The contents of the flask were stirred occasionally until the temperature was rose to approximately 37 "C whereupon @02 evolved and the yeast started to liquefy. The mixture was kept at 37 "C for 2 h with occasjonal stirring, cooled to 4 "C, and 180 ml of cold 0.01 M Trisacetate, pH 8.5, were added. After 3 h at 4 "C, the extract was siphoned from below the emulsion and the pH maintained at 7.5 by addition of 1 M Trisacetate buffer, pH 8.5. Protease inhibitor was added and the suspension centrifuged at 10 000 rpm in a Sorvall RC 2-B centrifuge for an hour at 4 "C. The resulting supernatant (200 ml) was removed. A small aliquot was removed for dialysis and activity assay, and the remainder immediately subjected to ammonium sulfate fractionation. Step 2: Arnmonium Sulfate FracEionabion Solid ammonium sulfate was added with stirring over a period of 30rnin to 50% saturation at 4 "C. Stirring was continued for an additional 30 min before centrifugation (10 000 rpm for 1 h). The supernatant was adjusted to 70% saturation by the gradual addition of solid ammonium sulfate. After stirring for another 30 min, the suspension was centrifuged at 10 000 rpm for B h. The precipitate obtained was dissolved in a small volume ( 18 ml) of 10 mM potassium phosphate (pH 7.2)' 5 mikf mercaptoethanol, 1 mild EDTA, and 10% propylene glycol, then PMSF was added and the solution was dialyzed with three changes of the same buffer to remove excess salt. Step 3: DEAE-cellulose Chromatography Preparation of Column DEAE-cellulose was suspended in 2.0 M ammonium carbonate poured into a column with a diameter of 2.5 cm, and packed to a height of 30 cm. It was washed
10
20 FRACTIONS
30
FIG. 1, Chromatographic purification of ammonium sulfate fraction on a column of DEAE-cellulose in the presence of 35:& (v/v) propylene-glycol as described in Materials and Methods. Azsoand radioactivity are represented by closed and open circles, respectively. with 2 M ammonium carbonate until the optical density of the eluates at 260 nm was zero, then with several bed volumes of 0.1 M ammonium carbonate containing 0.1 M EDTA and finally with water until the eluate was neutral. The cellulose was converted to the phosphate form with 0.5 M potassium phosphate (pH 7.2) and then washed with 0.08 M potassium phosphate buffer (pH 7.2) containing 5 mM mercaptoethanol, 1 mM EDTA, 35% propylene gIycol, and protease inhibitor (500 pg/100 ml solution). The dialyzed product from step 2 was then applied and the column washed with 50 an1 of the above buffer. Elution was carried out with a linear gradient from 80 to 320 mM potassium phosphate (pH 7.2), both buffers containing 5 mM mercaptoethanol, 1 mM EDTA, 35% propylene glycol. and PMSF inhibitor. Flow rates were maintained at approximately 1 ml/min. Fractions of 20 ml were collected. Enzymic activity was eluted at a phosphate concentration of 0-1 M (Fig. 1). Fractions containing PRS3 activity were pooled (I00 ml) . Step 4: Hydroxylapatite Chronzatography The pooled fractions were applied to a 3.2 cm x I0 cm column of hydroxylapatite and powdered cellulose (4: I, w/w ) equilibrated with 10 mM potassium phosphate (pH 7.2), 5 mM mercaptoethanol, 1 mM EDTA, 20% propylene glycol, and PMSF. After application of the sample, the column was eluted with a linear gradient of 180-300 ml potassium phosphate (pH 7.21, both buffers containing 5 mM mercaptoethanol, l mM EDTA, 20% propylene glycol, and PMSF. The bulk of the protein elutes early while the enzj7meappears approximately half way in the gradient (Fig. 2). The active fractions (50 ml) were pooled, diluted with 2 volumes of H20, stirred gently and applied to a hydroxylapatite column (2 m x 3 cm); elution was carried out with a minimum volume of 0.5 Ad potassium phosphate (pH 7.2), 5 mM EDTA, and 10% propylene glycol. This step was suggested by Dr. B. Reid, as giving greater yields than over the final ammonium sulfate step of Reid et al. (12). The purified enzyme was either used for analysis or stored at
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HOSSAIN: YEAST
PHENYLALANYL-tRNA SYNTHETASE
FRACTIONS
FIG.2. Gradient elution of BEAE-cellulose fraction on a column of hydroxylapatite by the procedure outlined in Materials and Methods. A280 and radioactivity are represented by closed and open circles, respectively. TABLE1. Purification of Phe-tRNA synthetase (PRS) Fraction 1. 2. 3. 4.
Post-mitochondria1supernatant (Nfi)aSOr fraction DEAE-cellulose fraction Hydrsxylapatite fraction (final enzyme)
Protein (mg)
Specific activity (cpm/rng protein)
1480 674 12.4 0.5
0.003 0.005 0.12 2.1
X 10' X 10V X106 X 106
Total activity 4.8 X 10" . 3 7 X lo6 1.488X106 1.05 X 106
Recovery Purification
(5%)
1 1.67 40
100 69.9 30.9 21.7
700
TABLE 2. Proteolytic contamination as assayed by the casein digestion method at various stages sf purification Sample 1. 2. 3. 4.
Post-mitochondria1supernatant (NIf4)2S04fraction BEAE-cellulose fraction Hydroxylapatite fraction
~ ~ ~ of ~ protein / m g
Azsr/mg of protein
Ar2a/mg of protein
0.243 0.203 0.008 0 .MI8
Proteolytic Activity of the Enzyme Fractions Proteolytic enzyme activity in the extracts at each stage of purification was determined on the Kinetic Measurements Activity of PRS was assayed by measuring the casein substrate by the methods outlined in incorporation of L-rPC]phenylalanine into tRNAPfae 'Materials and Methods'. The results in Table 2 as described earlier in this text. Initial velocities were obtained using concentrations which ensure a linear indicate that this extraction procedure leads to relationship between reaction rates and incubation greatly diminished (although measurable) time. All kinetic constants were fitted to data by a levels of proteolytic activity in the early phases; numerical procedure of Dr. Richard Viale, using a in later stages no proteolytic activity can be dePDP-10 computer. The rate patterns were deduced tected on the casein substrate used. from inspection of Lineweaver-Burk reciprocal plots constructed from the numerically derived constants. Assessment o f Purity and Determination o f Typical experiments were run for severd fixed conMolecular Weight centration values of a second reactant in the presence The purified enzyme obtained after hydroxsf a saturating concentration of a third reactant. ylapatite chromatography was subjected to analysis by gel filtration and electrophoresis. Results Figure 3 shows the elution profile obtained by Puri fic~tion chromatography on a column of Bio-Gel A The phenylalanine-tRNA synthetase (PRS ) (1-5 m ) using buffers containing 0.5 M ammohas been purified 700-fold. nium sulfate. The preparation appears to be
-20 after mixing with an equal volume of propylene glycol. OC.
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CAN. J .
BHOCHEM. VOL. 53.
1975
FRACTIONS
FIG.3. Gel filtration of purified PRS. Purified enzyme (2.5 mg)in 3 ml of 10 m M potassium phosphate (pH 7.2), 5 mM mercaptoethanol, 1 mM EDTA, and 1O& : glycerol was applied to a column (I X 78 cm) of Bio-Gel A (8.5 m) equilibrated in the same buffer containing 0.5 M ammonium sulfate. Fractions of 1.5 ml were collected, monitored for absorbance at 280 nm, and assayed for PRS activity as described in the text. The arrows refer to the elution positions of (I) blue dextran (BD) (rnol. wt. 2 X 80"; (2) rabbit muscle pyruvate kinase (PM); (3) lactic dehydrogenase (LDM) (msl. wt. 150 088); and (4) ovalbumin (QA) (mol. wt. 43 OW).
FIG.5. Electrophoresis of purified PRS in polyacrylamide gels containing SDS and estimation of the mol. wt. of its subunits. The enzyme was dissociated into subunits and subjected to the conditions described in Materials and Methods; 10pg of enzyme used. The molecular weight of the protein markers are plotted, on a logarithmic scale, against their mobilities on acrylamide gels during electrophoresis in the presence of SDS. The proteins are pgalactosidase (@-gal,130 00); serum albumin (SA, 68 W); ovalbumin (CIA, 43 000); and DNA* (31 000).
data according to the method of Andrew (24) as shown in Fig. 4 yields a value sf approximately 350 000. Sodium Dodecyl Sulfate (SDS) Gel EBectrophoretic Studies of PRS Purified PRS was dissociated into subunits in SDS-mercaptoethad and subjected t s elecFIG.4. Estimation of the molecular weight of PWS in trophoresis in pslyacrylamide gels containing Bio-Gel A (1.5 m) as described in the text. The molecular SDS using the method of Weber a d Bsborne weight of the protein markers are plotted, on a logarithmic ( 2 3 ) with the modifications described in 'Mascale, against their K a ,, the par tition coefficient between terials and Methods'. As in Fig. 5 , a single band the liquid phase and the gel phase. K,, = (V, - Vo)j was observed under standard electrophoretic (V, - Vo), in which V, is the elution volume of the sample, V, the total volume of the gel bed, and Vo the conditions. Calculation of the molecular weight void volume evaluated by blue dextran. Pyruvate kinase of this band by comparison to the relative mo(PM, mol. wt. 230 000 - 237 000); lactic dehydrogenase bilities yields a value close to 75 000. A second (LDH, rnol. wt. 150 OW); and ovalbumin (QA, mol. wt. subunit of 65 800 daltons has also been de43 m). tected sometimes similar to that observed by Reid et al. ( I 2 1. quite homogeneous as evidenced by a single protein peak of constant specific activity. Also Initial Velocity Studies shown in Fig. 3 are the elution positions of An intersecting rate pattern was observed known msIecular weight standard proteins after when %/v was plotted against 1/[Phe] for exchromatography on the same column. Estima- periments done at different fixed Bevels of ATP tion of the molecular weight of PRS from these (Fig. 6A). A family sf lines intersecting to the
HOSSAIN: YEAST PMBNYLALANYL-tRNA SYNTHETASE
Al - fixed ATP
l e ~ l s
I
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ATP
- Varied
i3
-
~ R N A fixed ~ ~ levels ATP
-
saturated levels
8 Phe
- SATURATED LEVELS
"0, 10
.,
FIG.6. Initial velocity patterns (A) with phenylalanine as the variable substrate at different fixed concentrations of 1.5 m M ATP); (B) with ATP as thevariable substrate at ATP at saturating levels of tRNAPhe ( e , 0-75 m M ATP; 0, 50 pM Phe; different fixed concentrations of phenylalanine at saturating levels of tRNAPhe ( e , 25 yM Phe; 8, 100 pM Phe); ( 6 )with phenyialanine as the variable substrate at different fixed concentrations of tRNA at saturating concentrations sf ATP ( e , 0.2 pM tRNAPhe; 8 , 0.5 pcMtRNAPhe; A, 1.8 pM tRNAPhe); (19) with ATP as the variable substrate at different fixed concentrations sf tRNA at saturating levels of phenylalanine ( e , 0.25 pM tRNA; 8 , 0.5 pM tRNA; 18 pM tRNA).
.,
left of the vertical axis was noted when l / v was plotted against B/[ATP] for experiments done with different concentrations of L-phenylalanine and with saturating levels of tRNAghe (Fig. 6B). From an inspection of Fig. 6 A and B, it can be seen that both the slope and the intercepts vary. This suggests that the variable and fixed substrate may combine with different enzyme forms, which are reversibly connected. According to Cleland (261, this pattern suggests a mechanism in which substrate ATP and phenylalanine binds to the enzyme before any product can be formed.
When phenylalanine was the variable substrate at several fixed concentrations of tWNAPb and saturating ATP concentrations, the double reciprocal plots exhibit a parallel pattern (Fig. 66.). This pattern also resulted when ATP was varied at different fixed concentrations of tRNAPhe (Fig. 6D). Non-intersecting patterns are consistent with a Ping-Pong mechanism in which both variable and changing fixed substrate combine with different enzyme forms not interconnected through reversible steps. The rouglaly linear patterns which were evaluated by an inspection of the slope and
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CAN. J. BIOCHEM.
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intercepts of Fig. 6 A and B probably suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion, but do not, however, completely eliminate a random mechanism. Discussion The toluene lysis procedure described above provides a means of isolating yeast P R S q n a highly active form. In essence, our procedure represents a hybrid between that applied by von Tigerstrom and Tener ( 17) to the yeast histidyl-tRNA synthetase (EC 6.1.1.21 ) (partially purified) and the very successful preparation method for PRS due to Reid et al. ( 12). In particular, we have availed ourselves of a modification of the last step in this latter procedure due to Dr. Reid. The most obvious explanation which accounts in a general way for the variability among the published accounts of these enzymes is the presence of proteolytic activity in the yeast. Commercial preparations of yeast hexokinase (EC 2.7.1.1 ) , for example, have been found to be contaminated with traces of proteolytic activity which has complicated determination of the size of the polypeptide subunit of this enzyme (25). Assay of proteolysis using casein indicates that even in the presence of phenylmethylsulfonyl fluoride and following toluene lysis some activity remains (see Table 2). Nevertheless, the present procedure offers a route to reliable preparation of the enzyme on a large scale, as would be essential for the preparation of crystals or detailed investigation of the interaction between the enzyme and the tRNAP". For a three substrate, three product reaction (ter-ter mechanism) there exist six possible mechanisms which may be either sequential or Ping-Pong. Aminoacyl-tRNA synthetases reported so far demonstrate both kinetic patterns (25-27). While the results of this investigation with PRS are superficially consistent with an ordered binding process with ATP first, the actual mechanism cannot be established unequivocally by steady-state kinetic experiments and may be considerably more complex. The investigation by fluorescence equilibrium measurements of the interactions of PRS with its ligands will be reported in detail elsewhere.
VOL. 53.
1975
1. Raj Bhandary, U. L. & Chang, S. H. (1968) J. Biol. Chem. 243, 598-668 2. Philipsen, P., Thiebe, R., Wintermeyer, W.& Zachau, H. G . (1968) Biocherem. Biophys. Res. Commm. 33, 922-926 3. Suddath, F. L., Quigley, G. L., McPherson, A., Sneden, D., Kim, J. J., Kim, S. H. & Rich, A. (1974) Nature (Eondon) 248, 20-24 4. Kim, S. H., Sussman, J. L., Suddath, F. L., Quigley, G. J., McPherson, A., Wang, A. H. J., &man, N. 6. & Rich, A. (1974) Proc. Natl. Acud. Sci. U.S.A. 71, 4970-4974 5 . Rskrtus, J. D., h d n e r , J. E., Finch, J. F., Rhoda, D., Brown, R. D., Clark, B. F. C. & Klug, A. (1974) Nature (Latadon) 250, 54.6-551 6. Romer, R., Reisner, D. & Maass, G. (1970) FEBS b t t . 10, 352-357 7. Blum, A. D., Uhlenbeck, 8.D. & Tinms, I., Jr. (1972) Biochemistry 11, 324-3256 8. Cramer, F., Dmpner, H., vonder Waar, F., Schlimme, E. & Seidel, H. (1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1384-1391 9. Pongs, O., Bald, R. & Reinwald, E. (1973) Eur. J. Bioehem. 32, 117-125 10. Thiebe, R., H a r k n , K. & Zachau, H. G. (1972) Eur. J. Bioehem. 26,132-143 11. Fasiolo, F., Befort, N.,Boulanger, Y. & E k l , J. P. (1978) Biochim. Biophys. Actu 21 7, 305-3 18 12. Schmidt, J., Wang, R., Stanfield, S. & Reid, B. R. (197 1) Biochemistry 10, 3264-3268 13. Rue, B., Surover, M. & Dudock, D. (1973) Biochemistry 12, 41464154 14. Lazarus, N. R., Ramel, A. W., Rustum, Y. M. & Barnard, E. A. (1966) Biochemistry 5, 4003-4025 15. Lagerkvist, U. & Waldenstrom, J. (1964) J. Biol. 8, 28-37 16. Makrnan, M. H. & Cantoni, G . L. (1965) Biochemktry 4,1434-1442 17. von Tigerstrom, M. & Tener, G . M. (1967) C m . J . Biochem. 45, 1067-1074 18. Chulmecka, V., von Tigerstrum, M., Obrenan, P. D. gL Smith, C. J, (1969) J. B i d . Chem. 244, 5481-5488 19. Hossain, A. & Kallenbach, N.R. (1974) FEBS Lett. 45, 282-285 20. Lowry, 0. H., R~senbrough,N. J., Faar, A. L. Br Randal, R. J. (1951) J. Biol. Chem. 193, 265-271 21. hskowsky, M. (1956) Metho& E~mzymol.2, 33-34 22. Schmidt, J. & Reid, B. R. (1971) Anal. Bioehem. 39, 162-1 69 23. Weber, K. & Osborn, M. (1969) J. Biol. Cltem. 244, 44064412 24. Andrews, B. (1965) Bioehcm. J. 96, 595-6435 25. Pringle, J. R. (1978) Biochem. Biophys. Res. Commun.
39,4650 26. Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-1 37 27. Penneys, N. S. & K. H. Muench (1974) Biochemistry 13, 54Q-571 28. Papas, T. S. & Peterkofsky, A. (1972) Biochemistry 11, 4602-4608 29. Papas, T. S. & Mehler, A. H. (1971) J. Biol. CItem. 246,5924-5928 30. Santi, D. V., Danenberg, P. V. & Satterly, P. (1971) Biochemistry 18, 4804-4820
