- Email: [email protected]
The isolation of a tryptophan-activating enzyme from pancreas
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66,
21-38 (1956)
The Isolation of a Tryptophan-Activating Enzyme from Pancreas’* 2 Earl W. Daviet Victor V. Koningsbergefi and Fritz Lipmann From the Biochemical Research Laboratory, Massachusetts General Hospital, and the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts Received June 25, 1956
In the course of attempts to study protein synthesis in cell-free prepaof pancreas, it was found that the particle-free supernatant of pancreas homogenate contained high concentrations of amino acidactivating enzymes of the type recently described by Hoagland et al. (1, 2) and by DeMoss and Novelli (3). These enzymes carry out the over-all reaction: rations
ATP + amino acid + hydroxylamine
+
aminoacyl hydroxamate
+ AMP + PP,
as well as an amino acid-dependent exchange resulting in the incorporation of radioactive PP into ATP. The highest activities were found with tryptophan as substrate. The tryptophan-activating enzyme soon showed promise for obtaining it in a rather high degree of purity. In 1 The following abbreviations will be used: ATP, adenosine triphosphate; AMP, adenosine monophosphate; GTP, guanosine triphosphate; GDP, guanosine diphosphate; GMP, guanosine monophosphate; PP, inorganic pyrophosphate; Pi , orthophosphate; PPase, pyrophosphatase; Tris, tris(hydroxymethyl)aminomethane. f This investigation was supported in part by a research grant from the National Cancer Institute, National Institutes of Health, United States Public Health Service; and the Life Insurance Medical Research Fund. 8 Fellow of the National Foundation for Infantile Paralysis; present address: Department of Biochemistry, Western Reserve University, Cleveland 6, Ohio. 4 Present address : Van’t Hoff Laboratory, University of Utrecht, Utrecht, The Netherlands. 21
22
EARL W. DAVIE, VICTOR V. KONINGSBERGER AND FRITZ LIPMANN
the following, the purification and characterization of this enzyme will be described. MATERIALS
AND METHODS
Salt-free hydroxylamine was prepared by the method of Beinert et ~2. (4). Its concentration was determined calorimetrically (5). Titration assay usually gave 10-150/ohigher values. Hydroxylamine concentration, in these experiments, refers to calorimetric values. Tryptophan hydroxamic acid was prepared by the method of Safir and Williams (6). It was used as calorimetric standard for the other amino acids as well as for tryptophan. The amino acids and peptides were purchased from the Nutritional Biochemical Corporation. Tryptazan was generously supplied by Dr. H. R. Snyder of the Department of Chemistry, University of Illinois. This compound gave a single ninhydrin spot when subjected to paper chromatography. The crystalline pyrophosphatase was kindly given to us by Dr. M. Kunita. Pa* was obtained from the Oak Ridge National Laboratories. The PPz* was prepared by pyrolysis of KHaP**Or ; it contained l-2% orthophosphate. Pyrophosphate was measured as orthophosphate by the method of Fiske and Subbarow (7) after acid or enzymatic hydrolysis. Radioactivity was determined by wet-counting samples on steel planchets. Protein concentrations were determined turbidimetrically with trichloroacetic acid using crystalline bovine serum albumin as a standard (8). GTP, GDP, GMP, and crystalline ATP were purchased from Sigma Chemical Company.
Hydroxamic Acid Assay; Enzyme Unit The method was similar to that used by Hoagland, Keller, and Zamecnik (2). Enzyme was routinely incubated with and without amino acid substrate at 37”, pH 7.8 in a reaction mixture containing lOpmoles ATP, 10pmoles MgClz , 10pmoles amino acid, 1OOOpmolessalt-free NHaOH, lOOpmoles Tris buffer, and 1Opg. crystalline pyrophosphatase/ml. Pyrophosphatase is essential because of strong product inhibition (cf. Fig. 7 and Table VI). One-milliliter aliquots were removed at appropriate times and added to 2.3 ml. of a solution containing 0.37 M FeCIS ,0.31 M trichloroacetic acid, and 0.65 M HCl for the determination of the hydroxamic acid according to Lipmann and Tuttle (9). One unit is that amount of enzyme which forms 1 pmole of tryptophan hydroxamate per hour in the standard assay. Specific activity is expressed as micromoles hydroxamate formed/mg. protein/hr. Proportionality between enzyme concentration and hydroxamate formation is shown in Fig. 1.
ATP-Pyrophosphate
Exchange
The purified salt-free enzyme was treated in a batch process with two portions of mixed-bed Dowex resin (equal portions of Dowex 50, Hf form, and Dowex 1, OH- form) for removal of residual amino acids. The treated enzyme was incubated with 3 pmoles ATP; 10 pmoles MgC12 ; 3 pmoles PP3z containing about 100,000 counts/mm., and 100 wales Tris buffer, pH 7.8 at 37”. Aliquots of the reaction
TRYPTOPHAN-ACTIVATINQ
ENZYME
23
EFFECT OF ENZYME CONC.
FICA 1. Assay contains : lOpmoles ATP, lOpmoles MgClt , 10 pmoles amino acid, 1000 pmoles NH,OH, 100 pmoles Tris buffer, 10 pg. pyrophosphatase, 20-170 pg. enzyme in 1.0 ml. Incubation at 37’, pH 7.8 for 15 min. mixture were added to 120/, trichloroacetic acid, and the PP and ATP were separated by charcoal adsorption according to the method of Crane and Lipmann (10). The results are expressed by an equation similar to that derived by DufEeld and Calvin (11) where: (ATP) (PP) 2.3 R 3 (ATP) + (PP) ’ t ’
log 100-s
100 exchange
R is the rate of exchange in ~moles/min., (ATP) and (PP) are the total concentrations of the reactants in amoles/ml.; t is time in minutes; and, 70 exchange =
ATP3,” x(ATPs; + PP”,” ’
loo’
where ATP:” and PP:’ refer to the total radioactivity in the ATP and PP at time t. This applies when, as in the present experiments, equimolar amounts of ATP and PP** were added. In this case, after equilibration, half of the added PP*’ is shifted into the ATP. ENZYME PREPARATION
AND PURIFICATION
From 8 to 10 lb. of beef pancreas, fresh from the slaughterhouse, is chilled in ice, freed of fat (2-3 lb.) and homogenized in 6 1. of ice-cold 0.15 M KCl, using a 4-l. Waring blendor. All steps in the purification are then carried out at O-4’. After a l-ruin. homogenization at full speed, the suspension is centrifuged for 5 min. at 5000 X g in a PR International centrifuge. The supernatant (5-6 1.) is passed through several layers of
24
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
cheesecloth to remove fat, and then 100-120 ml. of 1.0 M BaClz is added to give a final concentration of 0.02 M. The clumped microsomes are centrifuged off in 10 min. at 12,000 X g in a Servall centrifuge and discarded. The supernatant is again passed through cheesecloth for removal of residual fat to give Sup I. Isoelectric Precipitation Sup I is adjusted to pH 4.5 with 1.0 M acetic acid using a Beckman pH meter (previously standardized with buffer at room temperature) and immediately centrifuged at 5000 X g for 10 min. The precipitate is washed with about 10 vol. of 0.01 M acetate buffer, pH 4.5, containing 0.15 M KCl, recentrifuged, and frozen for overnight storage. After thawing, the precipitate is suspended in an acetate-KC1 solution containing 0.01 M N$304, recentrifuged, and extracted for about 1 hr. in 250 ml. of 0.15 M KC1 at a pH of 6.5. The pH is kept constant during the extraction by titration with 0.20 M NaOH. The suspension is centrifuged for 10 min. at 12,000 X g, giving Sup II. The precipitate is discarded. First Acid Fractionation The pH of Sup II is lowered to 5.2 with 0.20 M acetic acid followed by centrifugation for 10 min. at 12,000 X g; the precipitate is discarded. The pH of the supernatant is then further lowered to 4.8 and this precipitate is collected by recentrifugation, completely dissolved in 100 ml. of 0.15 KCl, and the pH is adjusted to 5.5 giving AC I. (NH&30~
Fractionation
An equal volume of ice-cold saturated ammonium sulfate containing 10 ml. of 4 M NaOH/l. is added to AC I, and the solution is centrifuged for 10 min. at 12,000 X g. The precipitate is discarded and the supernatant brought to 0.60 saturation by the addition of more (NH&S04 solution. If larger preparations are made, this fraction may be kept frozen in the presence of the (NH&SO1 which stabilizes the enzyme. Eventually the precipitate is dissolved in 4 ml. water and rapildy dialyzed for 2 hr. against ice-cold water to give Am 50-60. Second Acid Fractionation The pH of Am 50-60 is slowly lowered from about 6.5 to 5.3 by the addit,ion of 0.1 N acetic acid and centrifuged at 12,000 X g for 10 min.
TRYPTOPHAN-ACTIVATING
A Typical Puri$cation FriXtb
25
ENZYME
TABLE I Chart for a Batch of 7 Pounds of Beef Pancreas Micromoles hydrox/mg./hr.
Total units
0.40 38,000 Crude Pam., Sup I 4 26,700 pH 4.5-6.3, Sup II 8.8 19,400 pH 4.7-5.2, AC I 14,800 58 0.5-0.6 (NHJaSOh, Am 5@60 6,200 86 pH 4.5-5.0, AC II a This value varies from 0.3 to 1.5 in different preparations.
RWWWY
70 50 39 17
Similar precipitates are collected at pH’s 5.0 and 4.5. The first two precipitates and the final supernatant can be refractionated for recovery of additional enzyme. The pH 4.5 precipitate is dissolved in 2-3 ml. water and adjusted to about pH 6.5; this is fraction AC II. This preparation may be stored in the frozen state losing about 40 5%of its activity during 2 weeks’ storage. When salt-free solutions of preparation AC II are adjusted to about pH 4.8, large crystal plates form on freezing. Such crystals have the same activity as the mother liquid. Although such crystals were obtained rather regularly at this stage of purification, a routine method of crystallization still remains to be worked out. Table I shows a typical purification and recovery chart starting with 7 lb. of beef pancreas. Ultracentrifuge and Electrophoretic Analysis The ultracentrifuge studies were carried out with a Spinco analytical ultracentrifuge. Analyses were performed in 0.023 M phosphate buffer, pH 7.0, p = 0.15 after a 3-hr. rapid dialysis of an enzyme preparation. The preparations used still contained the nucleotide material described in a later paragraph in that the removal of this material has only more recently been possible. Figure 2 shows typical ultracentrifuge pictures of two enzyme preparations with specific activities of 58 and 80. Enzymatic activity was found to be associated with the major peak (75-80 % of the total protein) by comparing the two preparations of different specific activity. The per cent increase in the major peak was associated with the same increase in specific activity. Using a Yphantis-Waugh separation cell (12), the enzymatic activity was also found to be associated with the major peak. The authors wish to thank Drs. Yphantis and Waugh for their assistance in these experiments.
26
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
FIO. 2. Ultracentrifuge patterns for two different enzyme preparations with specific hydroxamate activities of 58 and 80. The top two pictures show the 68 enzyme after sedimentation of 66 and 88 min. at 59,780 r.p.m., 6.5” in 0.023 M phosphate buffer, pH 7.0 p = 0.15. Bottom two pictures show the 80 enzyme after 60 and 74 min. under identical conditions.
Preliminary electrophoretic analysis of the purest preparation in the same buffer likewise showed the presence of a major component comprising 70-80 % of the total protein in addition to several minor components. Presence and Release of Strongly Bound Nucleotides in Highly PuriJied Preparations The enzyme preparations of 50-80 units/mg. contained 34% of nucleotide material, measured by the 280-260 absorption ratio (13). This is not removed by Dowex 1 or charcoal, or by repeated isoelectric precipitation. However, it was found more recently that incubation with ATP, or better, ATP and tryptophan, releases the nucleotide material which then may be removed by isoelectric precipitation followed by charcoal t.reatment of the redissolved enzyme. For identification, perchloric acid extracts were subjected to paper electrophoresis in 0.04 M citrate buffer, pH 3.8. A main component was found which migrates like guanosine monophosphate (GMP) and is
!l’RYPTOPHAN-ACTIVATING
2.0 t
0
27
U.V. SPECTRUM OF NATIVE AND ATP- TRYPTOPHANE TREATED ENZYME
:: I.5 5 H 1.0 ;: 0 k
ENZYME
-- DIFFERENCE
0.5
230
250
270
220
310
WAVE LENOTH
330
(myI
Fro. 3. Enzyme in amount of 135 mg. was incubated 15 min. in the presence of 14 amoles ATP, 35 pmoles tryptophan, 20 pmoles MgClo , in final volume of 4.0 ml., pH 7.0. See text for further details.
further identified as a guanosine derivative by the blue fluorescence given in ultraviolet light after exposure to hydrochloric acid fumes. However, the characteristic shoulder of guanosine in acid solution was not seen in the absorption curve of the perchlorate extract. Such a shoulder at 278 ml.c appears, however, in the difference spectrum for enzyme before and after treatment with ATP and tryptophan as shown in Pig. 3. It is shown in Table II that neither hydroxamate formation nor pyrophosphate exchange depends on the presence of the tightly bound GMP-like compound. In view of the implication of GDP and GTP shown by the work of Keller and Zamecnik (14) as a catalyst in amino TABLE
II
Actiuity of Am 60-60 before and after Treatment with ATP and Tryptophan Hydroxamate assay: 10 amoles ATP, 10 rmoles MgCls , 1000 pmoles NHzOH, 100 rmoles Tris buffer, 10 amoles tryptophan, 0.10 mg. of enzyme, and 10 pg. pyrophosphatase in final volume of 1 ml. Incubation for 15 min. at 37”, pH 7.8. Exchange assay: 3 pmoles ATP, 10 pmoles MgCll ,3 amoles PPP2,l pmole tryptophan, 100 pmoles Tris buffer, pH 7.3,0.05 mg. enzyme. Incubation at 37”. Micromoles bydrox/mg./hr.
Original Treated
58 50
Micromoles PPn exchange/m&m.
300 290
28
EARL W. DAVIE, VICTOR V. KONINGSBERGER AND FRITZ LIPMANN EFFECT OF TRYPTOPHANE CONC. ON HYDROXAMATE FORMATION
f I Y)
<
0.6 t
0.4 JIM
0.6
1.2
TRYPTOPHANE
FIG. 4. Assayed under standard conditions
/ML.
as described under Methods.
acid incorporation, the strongly bound GMP in this preparation still remains of interest. Characte-risticsof the Hydroxamate Test The afllnity of the enzyme for tryptophan is high. The concentrationactivity curve of Fig. 4 indicates saturation at 0.5 pmole tryptophan/ml. under the conditions of these experiments. This saturation concentration is too high because at this and lower concentrations tryptophan was used up at the end of the experiment. In view of the limitations of the hydroxamate determination, however, a more exhausting analysis of the affinity for tryptophan was not attempted. The enzyme has likewise EFFECT OF ATP CONCENTRATION ON HYDROXAMATE
1.0
FORMATION
2.0
FICA 5. Assayed under standard conditions
3.0
as described under Methods.
TRYPTOPHAN-ACTIVATING
ENZYME
29
NH,OH CONC. t MOLES/L 1 FIG.
6. Assayed under standard conditions aa described under Methods.
a quite high aflinity for ATP, the ATP-activity curve of Fig. 5 showing saturation at 1.5 ccmoles ATP/ml. In spite of such a high affinity for the two primary reactants, in confirmation of the observations by Hoagland (1) and by DeMoss and Novelli (3), hydroxamate forms only in the presence of rather high concentrations of hydroxylamine. The experiment of Fig. 6 shows proportionality between hydroxamate formation and hydroxylamine concentration up to 3 M hydroxylamine. The primary reaction product resulting from the interaction of tryptophap and ATP, therefore, appears to react with hydroxylamine either only sluggishly or is not easily accessible to it. The reaction was routinely studied in 1 M NHtOH to avoid the neutralization of higher concentrations of base in the color test. Therefore, maximal activities represent values about threefold greater than those reported in the routine analysis. Phosphate Balance; Pyroplwsphate Inhibition The equivalence between hydroxamate and pyrophosphate formation was indicated in the experiments of Hoagland et al. (2) and it is confirmed here, as shown in Table III. Accumulation of pyrophosphate is more easily obtained with the present preparation, which does not contain any pyrophosphatase. However, during the incubation the reaction rate progressively decreases, while, in the presence of pyrophosphatase, the rate remains constant. The product inhibition by pyrophosphate is shown in Table IV, and its reversal through addition of pyrophosphatase is amptied by the experiment illustrated in Fig. 7. For unknown reasons,
30
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE III Comparison of Hydroxamate and Pyrophosphate OT Phosphate Formation Assay contains: 10 pmoles ATP, 10 pmoles MgCl? , 10 pmoles tryptophan, 1000 pmoles NHtOH, 100 pmoles Tris buffer and 0.08 mg. purified enzyme per 1 ml. reaction mixture. Incubation at 37”, pH 7.8. Ten micrograms crystalline pyrophosphatase/ml. added to the second reaction. Pyrophosphatase Time
+ Hydrox
10 20 30 40
0.43 0.69 0.86 1.0
PP
Hydrox
0.42 0.60 0.75 0.95
0.64 1.3 1.9 2.5
Pi/Z
0.59 1.2 1.8 2.5
TABLE IV Pyrophosphate Inhibition of Hydroxamate Formation Sample
Additions
1 2 3 4 5 6
PPase None 0.2 0.5 1.0 5.0
Mi~gnoesj . .
53 19 18 14 10 6
Per cent max. rate
100 36 34 26 19 11
AMP does not inhibit this reaction. Assay contains: 10 pmoles ATP, 10 amoles MgCll , 10 rmoles tryptophan, 1000 pmoles NHnOH, 100 amoles Tris buffer, O-5 amoles PP, 0.109 mg. purified enzyme in a final volume of 1.0 ml. Incubation 15 minutes at 37”, pH 7.8.
such a reversal was frequently rather incomplete with less pure enzyme preparations. It should be pointed out that AMP does not inhibit the hydroxamate reaction. The hydroxamate formed was identified as tryptophan hydroxamate by paper chromatography, using a butanol-acetic acid-water solvent system and confirmed through radioautography of the hydroxamic acid formed with CY4. The reaction is found to depend on the presence of magnesium when examined in the absence of pyrophosphatase. A more elaborate evaluation of the magnesium effect was not attempted in view of the magnesium dependence of pyrophosphatase, which is needed to maintain a constant rate.
TRYPTOPHAN-ACTIVATING
31
ENZYME
I
HYDROXAMATE FORMATION IN PRESENCE AND ABSENCE OF
6.0
-
20
40 TIME
60 60 IN MINUTES
100
Fra. ‘7. Assay contains: Curve A: 10 rmoles ATP, 10 pmoles MgCl~ , 10 pmoles tryptophan, 1000 rmoles NHsOH, 100 pmoles Tris, and 0.10 mg. enzyme/l ml. Incubation 37”, pH 7.8; 10 pg. pyrophosphatase/ml. added after 55 min. Curve B: same as A except 10 pg. PPase was added at zero time.
p-Chloromercuribenzoate Inhibit&n Although the addition of sulfhydryl compounds was not found to have a stimulatory effect even on the most highly purified preparation, the enzymatic reaction is strongly inhibited by p-chloromercuribenzoate, as shown in Fig. 8. Addition of 10 pmoles of cysteine reverses 100% inhibition approximately 30%. This partial reversal is due at least in part to an inhibition of the enzyme by higher concentrations of cysteine; p-chloromercuribenzoate has no effect on pyrophosphatase.
32
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
EFFECT 100 - #ERcuRl
4.2
AND
FRITZ
LIPMANN
OF PARR CHLORO BEfVZOfG ACfD
4.5 -Log
4.6 5.1 PCMB Cont.
5.4
FIQ. 8. Assay contains: 10 rmoles ATP, 10 pmoles MgClz , 10 pmoles tryptophan, 100 pmoles Tris buffer, 10 pg. PPase, and 0.10 mg. enzyme. Preincubated with various concentrations of PCMP in micromoles for 5 min. at 37”. Reaction started by the addition of 1000 pmoles NHzOH.
A TP-Pyrophosphate Amino acid-dependent pyrophosphate and ATP
exchange
Exchange between
radioactive
inorganic
had been used by previous workers alternatively with hydroxamate formation (l-3). A typical time curve for this reaction is shown in Fig. 9. It is of considerable interest now to compare the characteristics of the two types of reaction, using this nearly homogeneous enzyme preparation. It should be emphasized that the dependence of pyrophosphate exchange on tryptophan addition was only realized after the enzyme had been carefully treated with Dowex 50 to remove residual tryptophan. The most highly purified enzyme fraction was
1 0.6 -
TIME CURVE FOR PP32 EXCHANGE
4 8 12 TIME IN MINUTES
FICL 9. Assayed under standard conditions as described under Methods.
TRYPTOPHAN-ACTIVATING
Efect of Tryptophan
ENZYME
33
TABLE V Concentration on the Exchange Reaction
I-T%3han
Rate of exchange
None 0.001 0.005 0.025 0.100 0.300 1.00 5.00 10.0
15 200 380 430 430 440 420 230 190
Assay conditions: 3 rmoles ATP; 10 MmolesMgC12; 3 pmoles PP; 100pmoles Tris buffer, pH 7.8; 34 ag. enzyme; and various amounts of tryptophan/l ml. reaction mixture.
found still to contain approximately 1 mole of tryptophan/mole of enzyme protein assuming a molecular weight of around 20,000-30,000 for the enzyme. This tryptophan remains bound to the enzyme throughout the various fractionating procedures. As shown in Table V, the enzyme is saturated with about 0.005 rmole tryptophan/ml. and about half-saturated with 0.001 rmole/ml. It may be noted that in contrast to the hydroxamic acid reaction, higher concentrations of tryptophan inhibit pyrophosphate exchange while the higher pyrophosphate concentration has no effect on this reaction. The hydroxamate reaction, however, is 85 % inhibited by the 3 pmoles pyrophosphate/ml. used in this experiment (cf. Table IV). The higher afhnity for tryptophan with pyrophosphate exchange may be only apparent in view of the already mentioned limitations of the hydroxamic acid method. Of particular interest is the reactivity of tryptazan with the tryptophan-activating enzyme for hydroxamate formation as well as pyrophosphate exchange. The affiity for tryptophan is at least threefold that of tryptazan in the hydroxamate assay. The tryptazan inhibition of adaptive enzyme synthesis observed by Halvorson, Spiegelman, and Hinman (15) might therefore be due to erroneous incorporation rather than to an inhibition of tryptophan activation. We will report on tryptophan tryptazan competition for activating enzyme in a separate publication. The experiments with this nearly homogeneous enzyme preparation
34
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE VI Substrate Specificity Hydroxylambx
Substrate
Exchange
jm&?s/m&/hr.
n-Tryptophan nn-Tryptazan N-Acetyl-nn-tryptophan Glycyl-L-tryptophan n-Tryptophan n-Tyrosine L-+alanine n-Leucine Glycine n-Cysteine
jmoks/mg./hr.
400 64
86 48 0 0 0 3 0 0 0 0
-
8 8 0 0 0
Hydroxamate assay: 10 pmoles ATP, 10 amoles MgClz , 1000 amoles NHBOH, 100 amoles Tris buffer, 10 pmoles amino acid or peptide substrate, 0.10 mg. enzyme, and 10 pg. crystalline pyrophosphatase/ml. Incubation for 15 min. at pH 7.8.
PP” exchange assay: 3 moles ATP, 10 rmoles MgCll , 3 pmoles PPa*, 1 pmole amino acid, 100 pmoles Tris buffer pH 7.8, 0.04 mg. of Dowex-treated enzyme. Incubation at 37”.
support the proposition that hydroxamic acid formation and pyrophosphate exchange represent two aspects of the same enzymatic activity. This is amplified by the specificity of both reactions with regard to tryptophan and tryptazan shown in Table VI. It should be pointed out that the maximal activities for exchange and hydroxamate formation
a s ,f
300
I . i
200
1
I
I
I
I
6.0
7.0
8.0
9.0
I
PH FIG. 10. Assayed under standard conditions
as described under Methods.
TRYPTOPHAN-ACTIVATING
ENZYME
35
pH CURVE FOR TYROSINE HYDROXAMATE FORMAT ION
I
t
I
8.0
7.0
8.0
1
9.0
PH FIG. 11. Assayed under standard conditions
as described under Methods.
approach a similar value, as shown in Fig. 6, since the hydroxamate value increases about threefold on saturation with hydroxylamine. The marginal activity with tyrosine and phenylalanine is probably due to contamination with the respective activating enzymes which are present in the crude extracts in rather high concentrations. Further support for equivalence of the two reactions is given by the identical pH-activity curves of Fig. 10, with smoothly increasing activity up to pH 9. This curve differs from the pH-activity curve of Fig. 11 for tyrosine in the crude preparation, which has a sharp maximum at pH 7. Amino Acid Activation in Crude Extracts Qualitatively and quantitatively, the activities toward various amino acids appeared to be rather erratic with different extracts. Higher activities were found occasionally with methionine, while cysteine was usually nonreacting. As an illustration, some such results are presented in Table VII, showing a comparison also with the previous data of Hoagland et al. (2) recalculated to compare with our data. In the assay of the crude enzyme preparation there is a correction applied for a blank corresponding to about 0.05 pmole due to residual amino acids. This blank is completely absent in the purified preparation. We are inclined to believe that differences in activity toward the various amino acids may be due, at least in part, to differences in the characteristics and
36
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE VII Amino Acid Activation by Beef Pancreas5 Amino acid
Tryptophan Tyrosine Fhenylalanine Alanine Valine Glycine Cysteine Methionine Lysine Proline Leucine
Cm&pypn.
1.4 0.31 0.07 0.05 0.05 0.05 0 0.14 0.02 0.08 -
Dialyzed crude prepn. No. 1
2.1 0.24 0 0 0 0 0 0.38 0 0 -
Cmkpypn.
0.78 0.04 0.05 0.03 0.01 0.03 0 0.10 0.02 0.05 0.03
b
sup II
3.7 0.90 0.40 0.03 0.03 0.02 0.02 -
li%
in$te
0.11
0.07 0.01
0.03 0.10
0 Specific activity in micromoles hydroxamate/mg. protein/hr. b Data of Hoagland et al. (2) refer to 1 mg. liver fraction/ml. reaction mixture.
stability of the various activating enzymes. We consider it likely that with greater experience a complete spectrum of activating enzymes will be found. It is of interest that experiments kindly carried out by Dr. Keller show that pigeon pancreas supernatant can replace the liver supernatant used in the experiments of Hoagland, Keller, and Zamecnik (2) who presented evidence for a participation of the amino acid-activating liver supernatant in the process of amino acid incorporation into liver microsomes. COMMENTS
Possible Relation to Protein Synthesis A new type of ATP-linked condensation was introduced when it appeared that the ATP-acetate-CoA reaction (16), fatty acid activation in general (17, IS), and pantothenic acid synthesis (19) resulted in a pyrophosphate elimination and over-all liberation of AMP. The mechanism of this condensation was clarified through Berg’s demonstration that acyl adenylates react like intermediates in such systems (20). It earlier had been suggested that an analogous type of reaction may be involved in the amino acid activation for protein synthesis (21, 22). An important contribution toward the understanding of the mechanism of protein synthesis was made recently with the demonstration by Hoagland et al. (1, 2) of an ATP-linked amino acid activation with
TRYPTOPHAN-ACTIVATING
ENZYME
37
pyrophosphate elimination in liver extracts and the association of this system with the amino acid incorporation into liver microsomes of Keller and Zamecnik (23). DeMoss and Novelli (3), furthermore, could demonstrate the presence of such amino acid-activation systems in many microorganisms. They went a step further, by showing that leucyl adenylate behaves like an intermediary in this reaction (24). These experiments now indicate that an amino acyl adenylate may be an intermediary in peptide synthesis. This bears out an earlier proposition by Chantrenne (25), then working in Linderstrgm-Lang’s laboratory, that a substituted acyl phosphate would make a better precursor for peptide linking than the acyl phosphate itself. More recently, in his Lane Lectures, Linderstrem-Lang (26) again favored the general concept of a phosphoryl activation as a preliminary in protein synthesis, and it is with particular pleasure that we offer here a further support for such a contention as a contribution in a volume honoring Kaj Linderstr@m-Lang. Mechanism of Carboxyl Activation by Wuy of Pyrophosphate Elimination from A TP Although the reactivity of the acyl adenylate (20,24) with pyrophosphate is very remarkable, the forward reaction: ATP +RCOO-+AMP-COR
+PP
has so far been shown only in an indirect manner by using hydroxylamine in massive concentration as a trap for the activated acid. We have described here the isolation of a nearly homogeneous enzyme protein carrying out this type of reaction. We hope that soon the opportunity may arise to study such a reaction now with this enzyme as reactant. Such a possibility seems to promise some progress toward the fuller understanding of this seemingly important type of condensation. SUMMARY
The particle-free supernatant of pancreas homogenate contains enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. A great variety of ammo acids are activated and the extracts are particularly rich in tryptophan-activating enzyme. This enzyme was isolated and a fraction was obtained which contained approximately 70-80 % enzyme protein, as judged from ultracentrifugation and electrophoretic data.
38
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERQER
AND
FRITZ
LIPMANN
Enzyme activity is measured by tryptophan-specific hydroxamate formation and may also be tested for by tryptophan-linked ATP pyrophosphate exchange. The only amino acid showing a comparable affinity with the nearly pure enzyme is tryptazan which, at saturation levels, is approximately half as active as tryptophan in the hydroxamate reaction. REFERENCES 1. HOA~LAND, M. B., Biochim. et Biophys. Acto 16, 228 (1955). 2. HOA~LAND, M. B., KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 218, 345 (1956). 3. DEMOSS, J. A., AND NOVELLI, G. D., Biochim. et Biophys. Acta 18,592 (1955). 4. BEINERT, H., GREEN, D. E., HELE, P., HIFT, H., VON KORFF, R. W., AND RAMAKRISHNAN, C. V., J. Biol. Chem. 203,35 (1953). 5. FREAR, D. S., AND BURRELL, R. C., Anal. Chem. 27, 1664 (1955). 6. SAFIR, S. R., AND WILLIAMS, J. H., J. Org. Chem. 17, 1298 (1952). 7. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 8. STADTMAN, E. R., NOVELLI, G. D., AND LIPMANN, F., J. Biol. Chem. 191, 365 (1951). 9. LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem. 163,571 (1944). 10. CRANE, R. K., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). 11. DUFFIELD, R. B., AND CALVIN, M., J. Am. Chem. Sot. 68, 557 (1946). 12. YPHANTIS, D., AND WAU~H, D., J. Phys. Chem. 60, 623, 630 (1956). 13. WARBUR~, O., AND CHRISTIAN, W., Biochem. 2. 310, 384 (1941). 14. KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 221, 45 (1956). 15. HALVORSON, H., SPIEQELMAN, S., AND HINMAN, R. L., Arch. Biochem. and Biophys. 66, 612 (1955). 16. JONES, M. E., BLACK, S., FLYNN, R. M., AND LIPMANN, F., Biochim. et Biophys. Acta 12, 141 (1953). 17. JENCKS, W. P., Federation Proc. 12,703 (1963). 18. MAHLER, H. R., WAEIL, S. J., AND BOCK, R. M., J. Biol. Chem. 204, 453 (1953). 19. MAAS, W. K., AND NOVELLI, G. D., Arch. Biochem. and Biophys. 43,236 (1953). 20. BERN, P., J. Am. Chem. Sot. 77,3163 (1956). 21. LIPMANN, F., Advances in Enzymol. 1, 100 (1941). 22. LIPMANN, F., in “Mechanism of Enzyme Action” (McElroy, W. D., and Glass, B., eds.), p. 599. Johns Hopkins Press, Baltimore, 1954. 23. KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 2O!l, 337 (1954). 24. DEMOSS, J. A., GENUTH, S. M., AND NOVELLI, G. D., Federation Proc. 16,241 (1966). 25. CEANTRENNE, H., Compt. rend. trav. lab. Curlsberg 26, 297 (1948). Lectures, Proteins and Enzymes.” 26. LINDERSTR~M-LANO, K., “Lane Medical Stanford Univ. Publ., Med. Sci., Vol. 6. Stanford Univ. Press, Stanford Univ., Calif., 1952.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66,
21-38 (1956)
The Isolation of a Tryptophan-Activating Enzyme from Pancreas’* 2 Earl W. Daviet Victor V. Koningsbergefi and Fritz Lipmann From the Biochemical Research Laboratory, Massachusetts General Hospital, and the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts Received June 25, 1956
In the course of attempts to study protein synthesis in cell-free prepaof pancreas, it was found that the particle-free supernatant of pancreas homogenate contained high concentrations of amino acidactivating enzymes of the type recently described by Hoagland et al. (1, 2) and by DeMoss and Novelli (3). These enzymes carry out the over-all reaction: rations
ATP + amino acid + hydroxylamine
+
aminoacyl hydroxamate
+ AMP + PP,
as well as an amino acid-dependent exchange resulting in the incorporation of radioactive PP into ATP. The highest activities were found with tryptophan as substrate. The tryptophan-activating enzyme soon showed promise for obtaining it in a rather high degree of purity. In 1 The following abbreviations will be used: ATP, adenosine triphosphate; AMP, adenosine monophosphate; GTP, guanosine triphosphate; GDP, guanosine diphosphate; GMP, guanosine monophosphate; PP, inorganic pyrophosphate; Pi , orthophosphate; PPase, pyrophosphatase; Tris, tris(hydroxymethyl)aminomethane. f This investigation was supported in part by a research grant from the National Cancer Institute, National Institutes of Health, United States Public Health Service; and the Life Insurance Medical Research Fund. 8 Fellow of the National Foundation for Infantile Paralysis; present address: Department of Biochemistry, Western Reserve University, Cleveland 6, Ohio. 4 Present address : Van’t Hoff Laboratory, University of Utrecht, Utrecht, The Netherlands. 21
22
EARL W. DAVIE, VICTOR V. KONINGSBERGER AND FRITZ LIPMANN
the following, the purification and characterization of this enzyme will be described. MATERIALS
AND METHODS
Salt-free hydroxylamine was prepared by the method of Beinert et ~2. (4). Its concentration was determined calorimetrically (5). Titration assay usually gave 10-150/ohigher values. Hydroxylamine concentration, in these experiments, refers to calorimetric values. Tryptophan hydroxamic acid was prepared by the method of Safir and Williams (6). It was used as calorimetric standard for the other amino acids as well as for tryptophan. The amino acids and peptides were purchased from the Nutritional Biochemical Corporation. Tryptazan was generously supplied by Dr. H. R. Snyder of the Department of Chemistry, University of Illinois. This compound gave a single ninhydrin spot when subjected to paper chromatography. The crystalline pyrophosphatase was kindly given to us by Dr. M. Kunita. Pa* was obtained from the Oak Ridge National Laboratories. The PPz* was prepared by pyrolysis of KHaP**Or ; it contained l-2% orthophosphate. Pyrophosphate was measured as orthophosphate by the method of Fiske and Subbarow (7) after acid or enzymatic hydrolysis. Radioactivity was determined by wet-counting samples on steel planchets. Protein concentrations were determined turbidimetrically with trichloroacetic acid using crystalline bovine serum albumin as a standard (8). GTP, GDP, GMP, and crystalline ATP were purchased from Sigma Chemical Company.
Hydroxamic Acid Assay; Enzyme Unit The method was similar to that used by Hoagland, Keller, and Zamecnik (2). Enzyme was routinely incubated with and without amino acid substrate at 37”, pH 7.8 in a reaction mixture containing lOpmoles ATP, 10pmoles MgClz , 10pmoles amino acid, 1OOOpmolessalt-free NHaOH, lOOpmoles Tris buffer, and 1Opg. crystalline pyrophosphatase/ml. Pyrophosphatase is essential because of strong product inhibition (cf. Fig. 7 and Table VI). One-milliliter aliquots were removed at appropriate times and added to 2.3 ml. of a solution containing 0.37 M FeCIS ,0.31 M trichloroacetic acid, and 0.65 M HCl for the determination of the hydroxamic acid according to Lipmann and Tuttle (9). One unit is that amount of enzyme which forms 1 pmole of tryptophan hydroxamate per hour in the standard assay. Specific activity is expressed as micromoles hydroxamate formed/mg. protein/hr. Proportionality between enzyme concentration and hydroxamate formation is shown in Fig. 1.
ATP-Pyrophosphate
Exchange
The purified salt-free enzyme was treated in a batch process with two portions of mixed-bed Dowex resin (equal portions of Dowex 50, Hf form, and Dowex 1, OH- form) for removal of residual amino acids. The treated enzyme was incubated with 3 pmoles ATP; 10 pmoles MgC12 ; 3 pmoles PP3z containing about 100,000 counts/mm., and 100 wales Tris buffer, pH 7.8 at 37”. Aliquots of the reaction
TRYPTOPHAN-ACTIVATINQ
ENZYME
23
EFFECT OF ENZYME CONC.
FICA 1. Assay contains : lOpmoles ATP, lOpmoles MgClt , 10 pmoles amino acid, 1000 pmoles NH,OH, 100 pmoles Tris buffer, 10 pg. pyrophosphatase, 20-170 pg. enzyme in 1.0 ml. Incubation at 37’, pH 7.8 for 15 min. mixture were added to 120/, trichloroacetic acid, and the PP and ATP were separated by charcoal adsorption according to the method of Crane and Lipmann (10). The results are expressed by an equation similar to that derived by DufEeld and Calvin (11) where: (ATP) (PP) 2.3 R 3 (ATP) + (PP) ’ t ’
log 100-s
100 exchange
R is the rate of exchange in ~moles/min., (ATP) and (PP) are the total concentrations of the reactants in amoles/ml.; t is time in minutes; and, 70 exchange =
ATP3,” x(ATPs; + PP”,” ’
loo’
where ATP:” and PP:’ refer to the total radioactivity in the ATP and PP at time t. This applies when, as in the present experiments, equimolar amounts of ATP and PP** were added. In this case, after equilibration, half of the added PP*’ is shifted into the ATP. ENZYME PREPARATION
AND PURIFICATION
From 8 to 10 lb. of beef pancreas, fresh from the slaughterhouse, is chilled in ice, freed of fat (2-3 lb.) and homogenized in 6 1. of ice-cold 0.15 M KCl, using a 4-l. Waring blendor. All steps in the purification are then carried out at O-4’. After a l-ruin. homogenization at full speed, the suspension is centrifuged for 5 min. at 5000 X g in a PR International centrifuge. The supernatant (5-6 1.) is passed through several layers of
24
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
cheesecloth to remove fat, and then 100-120 ml. of 1.0 M BaClz is added to give a final concentration of 0.02 M. The clumped microsomes are centrifuged off in 10 min. at 12,000 X g in a Servall centrifuge and discarded. The supernatant is again passed through cheesecloth for removal of residual fat to give Sup I. Isoelectric Precipitation Sup I is adjusted to pH 4.5 with 1.0 M acetic acid using a Beckman pH meter (previously standardized with buffer at room temperature) and immediately centrifuged at 5000 X g for 10 min. The precipitate is washed with about 10 vol. of 0.01 M acetate buffer, pH 4.5, containing 0.15 M KCl, recentrifuged, and frozen for overnight storage. After thawing, the precipitate is suspended in an acetate-KC1 solution containing 0.01 M N$304, recentrifuged, and extracted for about 1 hr. in 250 ml. of 0.15 M KC1 at a pH of 6.5. The pH is kept constant during the extraction by titration with 0.20 M NaOH. The suspension is centrifuged for 10 min. at 12,000 X g, giving Sup II. The precipitate is discarded. First Acid Fractionation The pH of Sup II is lowered to 5.2 with 0.20 M acetic acid followed by centrifugation for 10 min. at 12,000 X g; the precipitate is discarded. The pH of the supernatant is then further lowered to 4.8 and this precipitate is collected by recentrifugation, completely dissolved in 100 ml. of 0.15 KCl, and the pH is adjusted to 5.5 giving AC I. (NH&30~
Fractionation
An equal volume of ice-cold saturated ammonium sulfate containing 10 ml. of 4 M NaOH/l. is added to AC I, and the solution is centrifuged for 10 min. at 12,000 X g. The precipitate is discarded and the supernatant brought to 0.60 saturation by the addition of more (NH&S04 solution. If larger preparations are made, this fraction may be kept frozen in the presence of the (NH&SO1 which stabilizes the enzyme. Eventually the precipitate is dissolved in 4 ml. water and rapildy dialyzed for 2 hr. against ice-cold water to give Am 50-60. Second Acid Fractionation The pH of Am 50-60 is slowly lowered from about 6.5 to 5.3 by the addit,ion of 0.1 N acetic acid and centrifuged at 12,000 X g for 10 min.
TRYPTOPHAN-ACTIVATING
A Typical Puri$cation FriXtb
25
ENZYME
TABLE I Chart for a Batch of 7 Pounds of Beef Pancreas Micromoles hydrox/mg./hr.
Total units
0.40 38,000 Crude Pam., Sup I 4 26,700 pH 4.5-6.3, Sup II 8.8 19,400 pH 4.7-5.2, AC I 14,800 58 0.5-0.6 (NHJaSOh, Am 5@60 6,200 86 pH 4.5-5.0, AC II a This value varies from 0.3 to 1.5 in different preparations.
RWWWY
70 50 39 17
Similar precipitates are collected at pH’s 5.0 and 4.5. The first two precipitates and the final supernatant can be refractionated for recovery of additional enzyme. The pH 4.5 precipitate is dissolved in 2-3 ml. water and adjusted to about pH 6.5; this is fraction AC II. This preparation may be stored in the frozen state losing about 40 5%of its activity during 2 weeks’ storage. When salt-free solutions of preparation AC II are adjusted to about pH 4.8, large crystal plates form on freezing. Such crystals have the same activity as the mother liquid. Although such crystals were obtained rather regularly at this stage of purification, a routine method of crystallization still remains to be worked out. Table I shows a typical purification and recovery chart starting with 7 lb. of beef pancreas. Ultracentrifuge and Electrophoretic Analysis The ultracentrifuge studies were carried out with a Spinco analytical ultracentrifuge. Analyses were performed in 0.023 M phosphate buffer, pH 7.0, p = 0.15 after a 3-hr. rapid dialysis of an enzyme preparation. The preparations used still contained the nucleotide material described in a later paragraph in that the removal of this material has only more recently been possible. Figure 2 shows typical ultracentrifuge pictures of two enzyme preparations with specific activities of 58 and 80. Enzymatic activity was found to be associated with the major peak (75-80 % of the total protein) by comparing the two preparations of different specific activity. The per cent increase in the major peak was associated with the same increase in specific activity. Using a Yphantis-Waugh separation cell (12), the enzymatic activity was also found to be associated with the major peak. The authors wish to thank Drs. Yphantis and Waugh for their assistance in these experiments.
26
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
FIO. 2. Ultracentrifuge patterns for two different enzyme preparations with specific hydroxamate activities of 58 and 80. The top two pictures show the 68 enzyme after sedimentation of 66 and 88 min. at 59,780 r.p.m., 6.5” in 0.023 M phosphate buffer, pH 7.0 p = 0.15. Bottom two pictures show the 80 enzyme after 60 and 74 min. under identical conditions.
Preliminary electrophoretic analysis of the purest preparation in the same buffer likewise showed the presence of a major component comprising 70-80 % of the total protein in addition to several minor components. Presence and Release of Strongly Bound Nucleotides in Highly PuriJied Preparations The enzyme preparations of 50-80 units/mg. contained 34% of nucleotide material, measured by the 280-260 absorption ratio (13). This is not removed by Dowex 1 or charcoal, or by repeated isoelectric precipitation. However, it was found more recently that incubation with ATP, or better, ATP and tryptophan, releases the nucleotide material which then may be removed by isoelectric precipitation followed by charcoal t.reatment of the redissolved enzyme. For identification, perchloric acid extracts were subjected to paper electrophoresis in 0.04 M citrate buffer, pH 3.8. A main component was found which migrates like guanosine monophosphate (GMP) and is
!l’RYPTOPHAN-ACTIVATING
2.0 t
0
27
U.V. SPECTRUM OF NATIVE AND ATP- TRYPTOPHANE TREATED ENZYME
:: I.5 5 H 1.0 ;: 0 k
ENZYME
-- DIFFERENCE
0.5
230
250
270
220
310
WAVE LENOTH
330
(myI
Fro. 3. Enzyme in amount of 135 mg. was incubated 15 min. in the presence of 14 amoles ATP, 35 pmoles tryptophan, 20 pmoles MgClo , in final volume of 4.0 ml., pH 7.0. See text for further details.
further identified as a guanosine derivative by the blue fluorescence given in ultraviolet light after exposure to hydrochloric acid fumes. However, the characteristic shoulder of guanosine in acid solution was not seen in the absorption curve of the perchlorate extract. Such a shoulder at 278 ml.c appears, however, in the difference spectrum for enzyme before and after treatment with ATP and tryptophan as shown in Pig. 3. It is shown in Table II that neither hydroxamate formation nor pyrophosphate exchange depends on the presence of the tightly bound GMP-like compound. In view of the implication of GDP and GTP shown by the work of Keller and Zamecnik (14) as a catalyst in amino TABLE
II
Actiuity of Am 60-60 before and after Treatment with ATP and Tryptophan Hydroxamate assay: 10 amoles ATP, 10 rmoles MgCls , 1000 pmoles NHzOH, 100 rmoles Tris buffer, 10 amoles tryptophan, 0.10 mg. of enzyme, and 10 pg. pyrophosphatase in final volume of 1 ml. Incubation for 15 min. at 37”, pH 7.8. Exchange assay: 3 pmoles ATP, 10 pmoles MgCll ,3 amoles PPP2,l pmole tryptophan, 100 pmoles Tris buffer, pH 7.3,0.05 mg. enzyme. Incubation at 37”. Micromoles bydrox/mg./hr.
Original Treated
58 50
Micromoles PPn exchange/m&m.
300 290
28
EARL W. DAVIE, VICTOR V. KONINGSBERGER AND FRITZ LIPMANN EFFECT OF TRYPTOPHANE CONC. ON HYDROXAMATE FORMATION
f I Y)
<
0.6 t
0.4 JIM
0.6
1.2
TRYPTOPHANE
FIG. 4. Assayed under standard conditions
/ML.
as described under Methods.
acid incorporation, the strongly bound GMP in this preparation still remains of interest. Characte-risticsof the Hydroxamate Test The afllnity of the enzyme for tryptophan is high. The concentrationactivity curve of Fig. 4 indicates saturation at 0.5 pmole tryptophan/ml. under the conditions of these experiments. This saturation concentration is too high because at this and lower concentrations tryptophan was used up at the end of the experiment. In view of the limitations of the hydroxamate determination, however, a more exhausting analysis of the affinity for tryptophan was not attempted. The enzyme has likewise EFFECT OF ATP CONCENTRATION ON HYDROXAMATE
1.0
FORMATION
2.0
FICA 5. Assayed under standard conditions
3.0
as described under Methods.
TRYPTOPHAN-ACTIVATING
ENZYME
29
NH,OH CONC. t MOLES/L 1 FIG.
6. Assayed under standard conditions aa described under Methods.
a quite high aflinity for ATP, the ATP-activity curve of Fig. 5 showing saturation at 1.5 ccmoles ATP/ml. In spite of such a high affinity for the two primary reactants, in confirmation of the observations by Hoagland (1) and by DeMoss and Novelli (3), hydroxamate forms only in the presence of rather high concentrations of hydroxylamine. The experiment of Fig. 6 shows proportionality between hydroxamate formation and hydroxylamine concentration up to 3 M hydroxylamine. The primary reaction product resulting from the interaction of tryptophap and ATP, therefore, appears to react with hydroxylamine either only sluggishly or is not easily accessible to it. The reaction was routinely studied in 1 M NHtOH to avoid the neutralization of higher concentrations of base in the color test. Therefore, maximal activities represent values about threefold greater than those reported in the routine analysis. Phosphate Balance; Pyroplwsphate Inhibition The equivalence between hydroxamate and pyrophosphate formation was indicated in the experiments of Hoagland et al. (2) and it is confirmed here, as shown in Table III. Accumulation of pyrophosphate is more easily obtained with the present preparation, which does not contain any pyrophosphatase. However, during the incubation the reaction rate progressively decreases, while, in the presence of pyrophosphatase, the rate remains constant. The product inhibition by pyrophosphate is shown in Table IV, and its reversal through addition of pyrophosphatase is amptied by the experiment illustrated in Fig. 7. For unknown reasons,
30
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE III Comparison of Hydroxamate and Pyrophosphate OT Phosphate Formation Assay contains: 10 pmoles ATP, 10 pmoles MgCl? , 10 pmoles tryptophan, 1000 pmoles NHtOH, 100 pmoles Tris buffer and 0.08 mg. purified enzyme per 1 ml. reaction mixture. Incubation at 37”, pH 7.8. Ten micrograms crystalline pyrophosphatase/ml. added to the second reaction. Pyrophosphatase Time
+ Hydrox
10 20 30 40
0.43 0.69 0.86 1.0
PP
Hydrox
0.42 0.60 0.75 0.95
0.64 1.3 1.9 2.5
Pi/Z
0.59 1.2 1.8 2.5
TABLE IV Pyrophosphate Inhibition of Hydroxamate Formation Sample
Additions
1 2 3 4 5 6
PPase None 0.2 0.5 1.0 5.0
Mi~gnoesj . .
53 19 18 14 10 6
Per cent max. rate
100 36 34 26 19 11
AMP does not inhibit this reaction. Assay contains: 10 pmoles ATP, 10 amoles MgCll , 10 rmoles tryptophan, 1000 pmoles NHnOH, 100 amoles Tris buffer, O-5 amoles PP, 0.109 mg. purified enzyme in a final volume of 1.0 ml. Incubation 15 minutes at 37”, pH 7.8.
such a reversal was frequently rather incomplete with less pure enzyme preparations. It should be pointed out that AMP does not inhibit the hydroxamate reaction. The hydroxamate formed was identified as tryptophan hydroxamate by paper chromatography, using a butanol-acetic acid-water solvent system and confirmed through radioautography of the hydroxamic acid formed with CY4. The reaction is found to depend on the presence of magnesium when examined in the absence of pyrophosphatase. A more elaborate evaluation of the magnesium effect was not attempted in view of the magnesium dependence of pyrophosphatase, which is needed to maintain a constant rate.
TRYPTOPHAN-ACTIVATING
31
ENZYME
I
HYDROXAMATE FORMATION IN PRESENCE AND ABSENCE OF
6.0
-
20
40 TIME
60 60 IN MINUTES
100
Fra. ‘7. Assay contains: Curve A: 10 rmoles ATP, 10 pmoles MgCl~ , 10 pmoles tryptophan, 1000 rmoles NHsOH, 100 pmoles Tris, and 0.10 mg. enzyme/l ml. Incubation 37”, pH 7.8; 10 pg. pyrophosphatase/ml. added after 55 min. Curve B: same as A except 10 pg. PPase was added at zero time.
p-Chloromercuribenzoate Inhibit&n Although the addition of sulfhydryl compounds was not found to have a stimulatory effect even on the most highly purified preparation, the enzymatic reaction is strongly inhibited by p-chloromercuribenzoate, as shown in Fig. 8. Addition of 10 pmoles of cysteine reverses 100% inhibition approximately 30%. This partial reversal is due at least in part to an inhibition of the enzyme by higher concentrations of cysteine; p-chloromercuribenzoate has no effect on pyrophosphatase.
32
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
EFFECT 100 - #ERcuRl
4.2
AND
FRITZ
LIPMANN
OF PARR CHLORO BEfVZOfG ACfD
4.5 -Log
4.6 5.1 PCMB Cont.
5.4
FIQ. 8. Assay contains: 10 rmoles ATP, 10 pmoles MgClz , 10 pmoles tryptophan, 100 pmoles Tris buffer, 10 pg. PPase, and 0.10 mg. enzyme. Preincubated with various concentrations of PCMP in micromoles for 5 min. at 37”. Reaction started by the addition of 1000 pmoles NHzOH.
A TP-Pyrophosphate Amino acid-dependent pyrophosphate and ATP
exchange
Exchange between
radioactive
inorganic
had been used by previous workers alternatively with hydroxamate formation (l-3). A typical time curve for this reaction is shown in Fig. 9. It is of considerable interest now to compare the characteristics of the two types of reaction, using this nearly homogeneous enzyme preparation. It should be emphasized that the dependence of pyrophosphate exchange on tryptophan addition was only realized after the enzyme had been carefully treated with Dowex 50 to remove residual tryptophan. The most highly purified enzyme fraction was
1 0.6 -
TIME CURVE FOR PP32 EXCHANGE
4 8 12 TIME IN MINUTES
FICL 9. Assayed under standard conditions as described under Methods.
TRYPTOPHAN-ACTIVATING
Efect of Tryptophan
ENZYME
33
TABLE V Concentration on the Exchange Reaction
I-T%3han
Rate of exchange
None 0.001 0.005 0.025 0.100 0.300 1.00 5.00 10.0
15 200 380 430 430 440 420 230 190
Assay conditions: 3 rmoles ATP; 10 MmolesMgC12; 3 pmoles PP; 100pmoles Tris buffer, pH 7.8; 34 ag. enzyme; and various amounts of tryptophan/l ml. reaction mixture.
found still to contain approximately 1 mole of tryptophan/mole of enzyme protein assuming a molecular weight of around 20,000-30,000 for the enzyme. This tryptophan remains bound to the enzyme throughout the various fractionating procedures. As shown in Table V, the enzyme is saturated with about 0.005 rmole tryptophan/ml. and about half-saturated with 0.001 rmole/ml. It may be noted that in contrast to the hydroxamic acid reaction, higher concentrations of tryptophan inhibit pyrophosphate exchange while the higher pyrophosphate concentration has no effect on this reaction. The hydroxamate reaction, however, is 85 % inhibited by the 3 pmoles pyrophosphate/ml. used in this experiment (cf. Table IV). The higher afhnity for tryptophan with pyrophosphate exchange may be only apparent in view of the already mentioned limitations of the hydroxamic acid method. Of particular interest is the reactivity of tryptazan with the tryptophan-activating enzyme for hydroxamate formation as well as pyrophosphate exchange. The affiity for tryptophan is at least threefold that of tryptazan in the hydroxamate assay. The tryptazan inhibition of adaptive enzyme synthesis observed by Halvorson, Spiegelman, and Hinman (15) might therefore be due to erroneous incorporation rather than to an inhibition of tryptophan activation. We will report on tryptophan tryptazan competition for activating enzyme in a separate publication. The experiments with this nearly homogeneous enzyme preparation
34
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE VI Substrate Specificity Hydroxylambx
Substrate
Exchange
jm&?s/m&/hr.
n-Tryptophan nn-Tryptazan N-Acetyl-nn-tryptophan Glycyl-L-tryptophan n-Tryptophan n-Tyrosine L-+alanine n-Leucine Glycine n-Cysteine
jmoks/mg./hr.
400 64
86 48 0 0 0 3 0 0 0 0
-
8 8 0 0 0
Hydroxamate assay: 10 pmoles ATP, 10 amoles MgClz , 1000 amoles NHBOH, 100 amoles Tris buffer, 10 pmoles amino acid or peptide substrate, 0.10 mg. enzyme, and 10 pg. crystalline pyrophosphatase/ml. Incubation for 15 min. at pH 7.8.
PP” exchange assay: 3 moles ATP, 10 rmoles MgCll , 3 pmoles PPa*, 1 pmole amino acid, 100 pmoles Tris buffer pH 7.8, 0.04 mg. of Dowex-treated enzyme. Incubation at 37”.
support the proposition that hydroxamic acid formation and pyrophosphate exchange represent two aspects of the same enzymatic activity. This is amplified by the specificity of both reactions with regard to tryptophan and tryptazan shown in Table VI. It should be pointed out that the maximal activities for exchange and hydroxamate formation
a s ,f
300
I . i
200
1
I
I
I
I
6.0
7.0
8.0
9.0
I
PH FIG. 10. Assayed under standard conditions
as described under Methods.
TRYPTOPHAN-ACTIVATING
ENZYME
35
pH CURVE FOR TYROSINE HYDROXAMATE FORMAT ION
I
t
I
8.0
7.0
8.0
1
9.0
PH FIG. 11. Assayed under standard conditions
as described under Methods.
approach a similar value, as shown in Fig. 6, since the hydroxamate value increases about threefold on saturation with hydroxylamine. The marginal activity with tyrosine and phenylalanine is probably due to contamination with the respective activating enzymes which are present in the crude extracts in rather high concentrations. Further support for equivalence of the two reactions is given by the identical pH-activity curves of Fig. 10, with smoothly increasing activity up to pH 9. This curve differs from the pH-activity curve of Fig. 11 for tyrosine in the crude preparation, which has a sharp maximum at pH 7. Amino Acid Activation in Crude Extracts Qualitatively and quantitatively, the activities toward various amino acids appeared to be rather erratic with different extracts. Higher activities were found occasionally with methionine, while cysteine was usually nonreacting. As an illustration, some such results are presented in Table VII, showing a comparison also with the previous data of Hoagland et al. (2) recalculated to compare with our data. In the assay of the crude enzyme preparation there is a correction applied for a blank corresponding to about 0.05 pmole due to residual amino acids. This blank is completely absent in the purified preparation. We are inclined to believe that differences in activity toward the various amino acids may be due, at least in part, to differences in the characteristics and
36
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERGER
AND
FRITZ
LIPMANN
TABLE VII Amino Acid Activation by Beef Pancreas5 Amino acid
Tryptophan Tyrosine Fhenylalanine Alanine Valine Glycine Cysteine Methionine Lysine Proline Leucine
Cm&pypn.
1.4 0.31 0.07 0.05 0.05 0.05 0 0.14 0.02 0.08 -
Dialyzed crude prepn. No. 1
2.1 0.24 0 0 0 0 0 0.38 0 0 -
Cmkpypn.
0.78 0.04 0.05 0.03 0.01 0.03 0 0.10 0.02 0.05 0.03
b
sup II
3.7 0.90 0.40 0.03 0.03 0.02 0.02 -
li%
in$te
0.11
0.07 0.01
0.03 0.10
0 Specific activity in micromoles hydroxamate/mg. protein/hr. b Data of Hoagland et al. (2) refer to 1 mg. liver fraction/ml. reaction mixture.
stability of the various activating enzymes. We consider it likely that with greater experience a complete spectrum of activating enzymes will be found. It is of interest that experiments kindly carried out by Dr. Keller show that pigeon pancreas supernatant can replace the liver supernatant used in the experiments of Hoagland, Keller, and Zamecnik (2) who presented evidence for a participation of the amino acid-activating liver supernatant in the process of amino acid incorporation into liver microsomes. COMMENTS
Possible Relation to Protein Synthesis A new type of ATP-linked condensation was introduced when it appeared that the ATP-acetate-CoA reaction (16), fatty acid activation in general (17, IS), and pantothenic acid synthesis (19) resulted in a pyrophosphate elimination and over-all liberation of AMP. The mechanism of this condensation was clarified through Berg’s demonstration that acyl adenylates react like intermediates in such systems (20). It earlier had been suggested that an analogous type of reaction may be involved in the amino acid activation for protein synthesis (21, 22). An important contribution toward the understanding of the mechanism of protein synthesis was made recently with the demonstration by Hoagland et al. (1, 2) of an ATP-linked amino acid activation with
TRYPTOPHAN-ACTIVATING
ENZYME
37
pyrophosphate elimination in liver extracts and the association of this system with the amino acid incorporation into liver microsomes of Keller and Zamecnik (23). DeMoss and Novelli (3), furthermore, could demonstrate the presence of such amino acid-activation systems in many microorganisms. They went a step further, by showing that leucyl adenylate behaves like an intermediary in this reaction (24). These experiments now indicate that an amino acyl adenylate may be an intermediary in peptide synthesis. This bears out an earlier proposition by Chantrenne (25), then working in Linderstrgm-Lang’s laboratory, that a substituted acyl phosphate would make a better precursor for peptide linking than the acyl phosphate itself. More recently, in his Lane Lectures, Linderstrem-Lang (26) again favored the general concept of a phosphoryl activation as a preliminary in protein synthesis, and it is with particular pleasure that we offer here a further support for such a contention as a contribution in a volume honoring Kaj Linderstr@m-Lang. Mechanism of Carboxyl Activation by Wuy of Pyrophosphate Elimination from A TP Although the reactivity of the acyl adenylate (20,24) with pyrophosphate is very remarkable, the forward reaction: ATP +RCOO-+AMP-COR
+PP
has so far been shown only in an indirect manner by using hydroxylamine in massive concentration as a trap for the activated acid. We have described here the isolation of a nearly homogeneous enzyme protein carrying out this type of reaction. We hope that soon the opportunity may arise to study such a reaction now with this enzyme as reactant. Such a possibility seems to promise some progress toward the fuller understanding of this seemingly important type of condensation. SUMMARY
The particle-free supernatant of pancreas homogenate contains enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. A great variety of ammo acids are activated and the extracts are particularly rich in tryptophan-activating enzyme. This enzyme was isolated and a fraction was obtained which contained approximately 70-80 % enzyme protein, as judged from ultracentrifugation and electrophoretic data.
38
EARL
W.
DAVIE,
VICTOR
V.
KONINGSBERQER
AND
FRITZ
LIPMANN
Enzyme activity is measured by tryptophan-specific hydroxamate formation and may also be tested for by tryptophan-linked ATP pyrophosphate exchange. The only amino acid showing a comparable affinity with the nearly pure enzyme is tryptazan which, at saturation levels, is approximately half as active as tryptophan in the hydroxamate reaction. REFERENCES 1. HOA~LAND, M. B., Biochim. et Biophys. Acto 16, 228 (1955). 2. HOA~LAND, M. B., KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 218, 345 (1956). 3. DEMOSS, J. A., AND NOVELLI, G. D., Biochim. et Biophys. Acta 18,592 (1955). 4. BEINERT, H., GREEN, D. E., HELE, P., HIFT, H., VON KORFF, R. W., AND RAMAKRISHNAN, C. V., J. Biol. Chem. 203,35 (1953). 5. FREAR, D. S., AND BURRELL, R. C., Anal. Chem. 27, 1664 (1955). 6. SAFIR, S. R., AND WILLIAMS, J. H., J. Org. Chem. 17, 1298 (1952). 7. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 8. STADTMAN, E. R., NOVELLI, G. D., AND LIPMANN, F., J. Biol. Chem. 191, 365 (1951). 9. LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem. 163,571 (1944). 10. CRANE, R. K., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). 11. DUFFIELD, R. B., AND CALVIN, M., J. Am. Chem. Sot. 68, 557 (1946). 12. YPHANTIS, D., AND WAU~H, D., J. Phys. Chem. 60, 623, 630 (1956). 13. WARBUR~, O., AND CHRISTIAN, W., Biochem. 2. 310, 384 (1941). 14. KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 221, 45 (1956). 15. HALVORSON, H., SPIEQELMAN, S., AND HINMAN, R. L., Arch. Biochem. and Biophys. 66, 612 (1955). 16. JONES, M. E., BLACK, S., FLYNN, R. M., AND LIPMANN, F., Biochim. et Biophys. Acta 12, 141 (1953). 17. JENCKS, W. P., Federation Proc. 12,703 (1963). 18. MAHLER, H. R., WAEIL, S. J., AND BOCK, R. M., J. Biol. Chem. 204, 453 (1953). 19. MAAS, W. K., AND NOVELLI, G. D., Arch. Biochem. and Biophys. 43,236 (1953). 20. BERN, P., J. Am. Chem. Sot. 77,3163 (1956). 21. LIPMANN, F., Advances in Enzymol. 1, 100 (1941). 22. LIPMANN, F., in “Mechanism of Enzyme Action” (McElroy, W. D., and Glass, B., eds.), p. 599. Johns Hopkins Press, Baltimore, 1954. 23. KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 2O!l, 337 (1954). 24. DEMOSS, J. A., GENUTH, S. M., AND NOVELLI, G. D., Federation Proc. 16,241 (1966). 25. CEANTRENNE, H., Compt. rend. trav. lab. Curlsberg 26, 297 (1948). Lectures, Proteins and Enzymes.” 26. LINDERSTR~M-LANO, K., “Lane Medical Stanford Univ. Publ., Med. Sci., Vol. 6. Stanford Univ. Press, Stanford Univ., Calif., 1952.