[124bl
ASPARTATE TRANSCARBAMYLASE FROM E. COLI
925
5-fluorocytidine28 Studies in man indicate that the inhibition by pyrimidine derivatives may be important in the regulation of pyrimidine synthesis by the cell.83-8~ 3. Hydrolysis o] Carbamyl~P. Extracts of a number of tissues 86 of a variety of vertebrates 87 decompose carbamyl-P enzymatically. An enzyme purifed 100-fold from brain is available, and it hydrolyzes both acetyl phosphate and carbamyl-PY The only other substrates tested were AMP, ATP, and phosphopyruvate, which were not attacked. No metal seems to be required, although Zn÷* and U02 ÷÷ inhibit. The pH optimum for carbamyl-P is 4, and for acetyl phosphate it is 5. The K~ values for the phosphates appear to be near 1 X 10-2 M. 4. Decomposition o] CitruUine. The arsenolysis of citrulline observed with rat liver extracts is thought to be due to OTC. 3~,88 Another activity observed in rat liver is the decomposition of citrulline, requiring ATP and AGA; the product is carbamyl-P. 89 5. Comparative Biochemistry. A recent comprehensive review of this subject is available2 ° L. H. Smith, Jr., and M. Sullivan, Biochim. et Biophys. Acta 39, 554 (1960). L. H. Smith, Jr., and F. A. Baker, J. Clin. Invest. 39, 15 (1960). ~J. A. Bain, Abstr. Papers of the 138t5 Meeting Am. Chem. Soc. p. 30c. New York (September, 1960). ~S. Grisolia and R. O. Marshall, Biochim. et Biophys. Acta 14, 446 (1954). ~S. Grisolia, J. Caravaca, and B K. Joyce, Biochim. et Biophys. Acta 29, 432
(1958).
~H. A. Krebs, L. V. Eggleston, and V. A. Knivett, Biochem. I. 59, 185 (1955L ~'L. H. Smith, Jr., and P. Reichard, Acta Chem. ~cand. I@, 1024 (1956). •op. p . Cohen and G. W. Brown, Jr., in "Comparative Biochemistry" (M. Florkin and H. S. Mason, eds.), Vol. If, p. 161. Academic Press, New York, 1960.
[ 124b]
Aspartate Transcarbamylase from
E~cherichia coli Aspartate ~ carbamyl phosphate --, Carbamyl aspartate ~ P~ ~- H +
By MARGARETSHEPHERDSON and ARTHUR B. PARDEE Assay Method The activity of this enzyme has been determined by several methods. The method used in the purification, 1 based on the estimation of inorganic phosphate, s is satisfactory provided phosphatase activity is 1 M. Shepherdson and A. B. Pardee, Y. Biol. Chem. 235, 3233 (1960). 2p. Reiehard and G. Hanshoff, Acta Chem. 8cand. 10, 548 (1956).
926
ENZrMES OF PROTEIN METABOLISM
[124b]
low. A sensitive but more time-consuming method involves the reaction of C14-aspartate with carbamyl phosphate, followed by determination of radioactivity of the carbamyl aspartate2 A third procedure is based on the colorimetric estimation of carbamyl aspartate. 4 The results are liable to considerable variation, and citrulline interferes. These two methods are useful with crude extracts of low activity. In a fourth method, the hydrogen ion produced is estimated in a pH-stat2 Reagents. Fifteen mieromoles of sodium aspartate, 8 micromoles of dilithium carbamyl phosphate (solution freshly prepared), and 50 micromoles of tris(hydroxymethyl)aminomethane (pH 7.0) are mixed in water to give a total of 1 ml. after the enzyme is added. The enzyme is dissolved in 0.01 M imidazole buffer (pH 7) containing 0.01 M mercaptoethanol (mercaptoethanol-imidazole buffer). Approximately 0.1 unit of enzyme is used per assay. Procedure. The substrate solution and enzyme are mixed and incubated at 28 ° for 20 minutes. At the end of the incubation period the reaction is stopped by immersing the tubes in ice water. Estimation of P~ in 0.1-ml. aliquots is carried out by the Lowry-Lopez method2 A blank containing the same components but omitting aspartate is included. The conditions are not optimal for the enzyme but assure a low blank. Definition of Unit and Specific Activity. A unit of enzyme activity is defined as the amount of enzyme which under the conditions of assay catalyzes the formation of 1 mieromole of P~ in 1 minute. Specific activity is expressed as units per milligram of protein. Protein determinations are carried out by the Folin method. T When mercaptoethanol is present in the samples it is necessary to include the same concentration in the controls, since it enhances the color produced by the reaction. Production of Enzyme Aspartate transcarbamlyase is a repressible enzyme. 4 The specific activity of the crude extract can be increased approximately a thousandfold if the bacteria are starved of pyrimidines before they are harvested. Bacterial Medium. The medium contains, per liter: 7 g. of K2HP04, 2 g. of KH2P04, 0.5 g. of sodium citrate • 5H20, 0.1 g. of MgS04 • 7H20, 1 g. of (NH4)2S04, 5 ml. of glycerol, 80 mg. of L-arginine, and 3.7 mg. of uracil. R. A. Yates and A. B. Pardee, J. Biol. Chem. 221, 743, 757 (1956). ' R. A. Yates and A. B. Pardee, J. Biol. Chem. 227, 677 (1957). sj. C. Gerhardt and A. B. Pardee, unpublished observation (1960). e O. H. Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946). ' O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 195, 265 (1951).
[124b]
ASPARTATE TRANSCARBAMYLASE FROM E. COLI
927
Bacterial Strain. A mutant of E. coli, R 185-482 (isolated by R. R. Roepke), that requires both arginine and uracil for growth is used. This organism lacks carbamyl phosphate synthetase? Growth of Bacteria. Twenty liters of medium are made up in a glass carboy with a capacity of just over 20 h The medium is inoculated to a density of 3 X 10~ cells/ml, with a culture of E. coil R185-482 which has been aerobically grown overnight at 37 ° in a medium containing 22 rag. of uracil per milliliter. The culture is incubated at 37 °, and aerobic conditions are maintained by passing moist, filtered air into the bottom of the carboy by means of two pieces of glass tubing inserted in the neck of the carboy through a loose cotton plug. Depending on the rate of aeration, the generation time of the bacteria is between 60 a n d l l 0 minutes. (There is some indication that the cultures yielding the richest source of enzyme are those with a longer generation time.) When a constant turbidity (about 4 X 10s cells/ml.) is reached, indicating utilization of all the uracil present in t h e medium, 4 g. of L-dihydroSrotic acid are added to the culture. Incubation is then continued for 5 to 12 hours, during which time the turbidity of the suspension approximately doubles. The specific activity of the enzyme increases for about 5 hours after addition of dihydroSrotic acid, after which it remains constant for at least a further 7 hours, although the total number of enzyme units present in the culture sometimes increases. The bacteria are harvested by Sharpies centrifugation either immediately after incubation or after storage overnight at 4 ° . Further steps are performed in the cold. The cream-colored bacterial mass obtained after centrifugation is suspended in about 50 ml. of water and subjected to sonic oscillation in a 10-kc. Raytheon magnetostriction oscillator at 4 ° for 20 minutes. The extract is centrifuged for 2 hours in the Servall centrifuge at 11,000 X g, and the precipitate, consisting of cell debris and unbroken cells, is discarded. The aspartate transcarbamylase activity of this extract was unchanged after storage for 3 months at --10% Data obtained at this and subsequent steps are shown in the table. Purification Procedure Considerable purification of t h i s enzyme has been achieved, commencing with repressed bacteria. 2 More recently a greater degree of purity was achieved by taking advantage of the large increase in specific activity of unrepressed bacteria.1 Step 1. Heat Fractionation.The cell-free extract obtained by sonic oscillation, adjusted to pH 6.0 with 0.5 M acetate buffer, is brought to 55 ° by swirling it in a flask immersed in a 58 ° water bath for about 8 M. Dirks and S. Grisolia, personal communication (1960).
928
ENZYMES OF PROTEIN METABOLISM
[124b]
3.5 minutes. After it is held at 55 ° for 5 minutes, the extract is cooled rapidly by swirling the flask in ice water. The heated extract is centrifuged at 11,000 X g for 30 minutes after which a fairly clear supernatant fluid is obtained. The precipitate is suspended in 10 ml. of water, brought to pH 6.0 with the acetate buffer, and recentrifuged; this supernatant fluid is added to the first. The enzyme activity in the heat-treated extract was stable for at least 2 months at --10°C. Step 2. Ammonium Sul]ate Fractionation. The supernatant fluid obtained after heat treatment is brought to pH 7 with 2 N N H 4 0 H and cooled in an ice-water bath. Solid (NH4)2S04 is added with stirring to give about 40% saturation; i.e., 243 mg./ml, of extract. After standing at 4 ° for not less than 3 hours the precipitate is centrifuged down, the supernatant fluid poured off, and the precipitate washed with about 5 ml. of 40% (NH4)2S04 at pH 7. After centrifugation the washing fluid is added to the main supernatant fluid. The supernatant fluid is readjusted to pH 7 with NH4OH, and more (NH4)~SO~ is added to bring the saturation to 50%, i.e., an additional 63 mg./ml, of supernatant fluid. Once again the preparation is centrifuged after standing for not less than 3 hours at 4 °. After centrifugation the precipitate is taken up in about 5 ml. of water. About 10% of the units originally present in the extract are found in the final supernatant fluid. Attempts to recover these units by addition of more (NH~)2SO~ were not very successful, as the specific activity of the precipitated material fell rapidly and the recovery of activity was poor. (The procedure has been successfully duplicated to this point by M. Derks and S. Grisolia.) It is necessary to remove most of the (NH,)2S04 from the dissolved 40 to 50% precipitate by dialysis before proceeding with the next step. Dialysis against water invariably results in complete inactivation of the enzyme, nor is much protection afforded by buffered solutions of thioglycolate, cysteine, or reduced glutathione. The enzyme is dialyzed, with stirring and in ½-inch dialysis tubing, overnight against 3 1. of mercaptoethanol-imidazole buffer. There is no loss of activity during dialysis, and the dialyzed material can be stored for at least 3 months at 4 °. On freezing the dialyzed preparation and rethawing it, a white precipitate appears and the activity is completely lost. After this stage, therefore, the enzyme preparation should be stored at 4 ° . Step 3. DEAE Fractionation. Before use, DEAE is washed by stirring 20-g. batches with four 1.5-1. volumes of 0.1 N KOH. The DEAE is then washed with water ~until the washing fluid is neutral. In the final wash the material which does not sediment after 20 minutes is discarded. The washed DEAE is collected under suction on a sintered-glass filter and dried at room temperature. Six grams of DEAE are made into a slurry
[124b]
ASPARTATE TRANSCARBAMYLASE FROM E. COLI
929
with water, allowed to settle by gravity in a 1-cm.-diameter column and equilibrated at 6 ° with mercaptoethanol-imidazole buffer, freshly prepared. The dialyzed extract obtained from 20 1. of culture is put on the column after being diluted 1 part to 5 of 0.01 M imidazole. Elution is started at a perfusion rate of 20 ml./hour with a 0.11 M KC1 solution made up in the mercaptoethanol-imidazole buffer. The eluate is collected in 10-ml. fractions, and the optical density of the fractions is followed at 260 m~ and 280 m~. A large amount of enzymatically inactive protein appears, after which the optical densities become fairly constant at a lower level. (The fractions following the peak sometimes began to show activity when rather large volumes, e.g., 280 ml., of the 0.11 M KC1 solution were put through the column.) Changing the eluant to 0.13 M KC1 in mercaptoethanol-imidazole buffer (pH 6.9) results in a protein peak which contains the enzyme. During the elution with 0.13 M KCI, 5-ml. fractions are collected. Gradient elution on DEAE has also been employed and found to be satisfactory (see also Reichard and Hanshoff2). In this case elution is begun with 0.1 M KC1 in mercaptoethanol-imidazole buffer in the mixing flask, and into this 1.0M KC1 in mercaptoethanol-imidazole buffer is slowly introduced, the rate of addition being the same as the rate of drainage of the column. Concentration o] the Enzyme. Those fractions in which the specific activity of the enzyme is constant and maximal, i.e., those under the 0.13M KC1 peak, are pooled (volume about 80 ml.) and diluted with 80 ml. of mercaptoethanol-imidazole buffer to lower the salt concentration. The diluted enzyme solution is then put on a 1-cm.-diameter column containing 0.6 g. of DEAE, prepared as before. The enzyme is completely retained by the column, no activity being detected in the effluent. Elution of the enzyme is accomplished with a 0.2 M KC1 in mercaptoethanol-imidazole buffer, and the eluate is collected in 2-ml. fractions. Recovery of the enzyme from this concentration step is almost 100%, and the activity is found entirely in the second to fourth, or third to fifth, fractions collected from the column. The specific activity remains unchanged. The fractions on either side of the enzyme peak have a specific activity approximately two-thirds that of the purer material. Subjecting this material to the same concentrating procedure results in a purification, the specific activity of the concentrated material being as high as that of the first concentrate. Some of the activity not recovered can be accounted for in the fractions following the 0.13M KC1 peak where the activity tails off
930
[124b]
ENZYMES OF PROTEIN METABOLISM
rather slowly. The purified enzyme dissolved iv mercaptoethanol-imidazole buffer was stable for a year at 4 °. Step 4. Crystallization. A portion of the enzyme preparation of specific activity about 117 and containing 13 rag. of protein per milliliter is adjusted to faint turbidity at room temperature by addition of a solution of saturated (NH4)~S0, made up in mercaptoethanolimidazole buffer. The final concentration of (NH,)2S04 is about 30% saturated. After about 2 weeks at 4 °, crystals in the shape of thin square plates should form. Excess (NH~)2SO~ is added to the bulk of the preparation, the precipitate is separated by centrifugation, taken up in a small volume of mercaptoethanol-imidazole buffer, brought to faint turbidity, and seeded with a small amount of the first crystals. Numerous crystals appear overnight from this solution. The material is readily recrystallized again by repetition of the above procedure. The specific activity of the two-times-crystallized material was only about 35. Presumably some denaturation had occurred during the period of standing in (NH,)2SO~. A summary of the purification procedure is given in the table. SUMMARY OF THE PURIFICATION PROCEDURE
Fraction
Extract Heat-treated extract 40-50% (NH~)~SO~precipitate DEAE fractions 1. Purest fraction 2. Less pure fraction (after refractionation)
Total Specific Enzyme Volume, enzyme, Protein, activity, yield, ml. units mg. units/rag. % 50 50 5
27,000 23,000 13,000
3280 1300 318
6
5,500
47
6
1,900
16
8 17 41
100 84 49
117 7 ~ 117
28
Properties
Physical Properties. The molecular weight is estimated to be 220,000, based on a sedimentation coefficient1 of 11.5 S. The net charge at pH 7 appears to be small and negative, because the enzyme has a low mobility at this pH on starch electrophoresis. 4 The purified material is homogeneous by both eentrifugation and electrophoresis criteria. Kinetic Properties. The turnover number under optimal conditions is about 100,000 molecules of substrate per minute. 1 The K~ is 4.5 X 10-~ M for carbamyl phosphate and 6 X 10-~ M for aspartate. The equilibrium at neutral pH lies very far in the direction of carbamyl aspartate formation? .B The optimum pH is 7.5, ~ or higher2
[125]
TttREONINE AND ALLOTHREONINE AI~DOLASE
931
Mechanism of Action. The two organic products are formed and two substrates disappear in approximately 1:1:1:1 ratios. Evidence is opposed to a carbamyl-enzyme intermediate and favors a single-displacement mechanism, z Inhibitors and Activators. The activity is 45% inhibited by 10-3 M p-Cl-mercuribenzoate.. This inhibition is reversed by glutathione and is prevented when both substrates are present. 2 A number of other common inhibitors are inactive toward this enzyme. 2 No activators of the enzyme are known. The enzyme is inhibited by various pyrimidinc derivatives of which cytidylic acid is the most potent (K1 about 6 X 10-3 M).5 This seems to be important in the regulation of pyrimidine synthesis by the cell.*
[ 125 ] Threonine and Allothreonine Aldolase CH~CHOHCHNH2COOH ~- CH3CHO -k CH2NH2COOH
By DAVID M. GREENBERG Since the discovery by Braunshtein and Vilenkina 1 of the aldol cleavage of/3-hydroxyamino acids, it has been determined that separate enzymes arc required for the different members of this group of amino acids and also for their allo and threo enantiomorphs. 2,3 Assay Method Acetaldehyde liberated in the reaction is trapped by diffusion in semicarbazide solution and determined colorimetrieally.
Reagents DL-threonine and DL-allothreonine, 0.5 M, adjusted to pH 7.6 with NaOH and stored in the cold. Pyridoxal phosphate, 10-3 M, dissolved in distilled water and stored frozen. Acetaldehyde, 0.2 M, prepared by dissolving 1.13 ml. of redistilled acetaldehyde in 100 ml. of distilled water. Semicarbazide reagent, 0.1876 g. of semicarbazide hydrochloride dissolved in 250 ml. of 0.15 M phosphate buffer, pH 7.6. Phosphate buffer, 0.15 M, pH 7.6. Prepared by dissolving 19.45 g. ~A. E. Braunshtein and G. Y. ¥ilenkina, Doklady Akad. Naulc. S.S.S.R. 66, 1243 (1949). 2 M. A. Karasek and D. M. Greenberg, J. Biol. Chem. 227, 191 (1957). 3 F. H. Bruns and L. Fiedler, Biochem. Z. 330, 324 (1958).