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[51] l -citramalate hydrolyase
[51]
L-CITRA.MALATE HYDROLYASE
331
[ 51 ] L- C i t r a m a l a t e H y d r o l y a s e
By C. C. WANQ and H. A. BARKER
coo-
I HO--C--CH3 L
CI~ _ COO L- (+) - C i t r a m a l a t e
-OOC~c/CH3 ~.
II
+
H20
H/C~.coo_ Mesaconate
A s s a y M e t h o d 1, 2
Principle. Citramalate hydrolyase activity is estimated by measuring the rate of absorbance increase at 250 n ~ resulting from the conversion of citramalate to mesaconate. The enzyme from Clostridium tetanomorphum consists of two separable protein components (I and II). The assay for each component is done by adding an excess of the other component. Maximal activity of the rate-limiting component in the assay is attained only after the two components have been incubated together ("activated") under carefully controlled conditions. Under suitable conditions the rate of absorbance increase is proportional to the concentration of the rate-limiting protein component.
Reagents Tris-HCI buffers, pH 8.2, 0.5 M and pH 8.4, 0.5 M 2-Mercaptoethanol (MET), 0.5 M. Prepare weekly Ferrous ammonium sulfate (FeAS), 10 mM. Prepare daily Tris buffer-cysteine-FeAS assay solution. This solution contains 60 mM Tris-HCl, pH 8.2, 2.0 mM L-cysteine hydrochloride, 2.0 mM NaOH, and 0.2 mM FeAS. It is made up in a graduate cylinder or similar vessel with a small air-surface to volume ratio and is mixed carefully to avoid excessive oxidation of cysteine. The temperature is adjusted to 25 °. The solution is allowed to stand at least 15 minutes; during this time the initial purple color fades (except at the surface) to give an almost color1A. It. Blair and It. A. Barker, J. Biol. Chem. 241, 400 (1966). 2C. C. Wang, Doctoral Dissertation, University of California, Berkeley, California,
1966.
332
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
less solution. The solution is stable for at least 4 hours when excessive contact with air is avoided DL-Citramalate, s disodium salt, 1.0 M, pH 7.0 Hydrolyase component I solution (for component II assay), 400 units per milliliter of 20 mM Tris-HC1 buffer, pH 7.0. The preparation should have a specific activity of at least 300 units per milligram of protein and should be free of component II activity Hydrolyase component I solution (for component I assay). The solution, diluted when necessary in 20 mM Tris-HC1 buffer, pH 7.0, should contain between 10 and 1000 units/ml Hydrolyase component II solution (for component I assay), 400 units per milliliter of 20 mM Tris-HCl buffer, pH 8.0, and 50 mM K2S04. The preparation should have a specific activity of at least 100 units per milligram of protein and should be relatively free of component I activity (less than 5 ~ of the component II specific activity) Hydrolyase component II solution (for component II assay). The solution, diluted when necessary in 20 mM Tris-HCl buffer, pH 8.0, and 50 mM K2S0~, should contain between 10 and 1000 units/ml Hydrolyase Component I Assay for Component I Procedure. The assay involves two distinct steps: (a) the "activation" of the system by incubating a rate limiting amount of component I with an excess of component II under specific conditions, and (b) the spectrophotometric assay proper, using an aliquot of the activated enzyme solution. The activation of the system is conveniently done in a reaction mixture having a volume of 100 pl, although a smaller volume may be used. On the I00 pl scale, 10 pl of hydrolyase component II solution (for component I assay) is added to 80 ~l of a freshly prepared solution (in a 0.5 ml test tube at 0 °) containing 10 pl 0.5M Tris-HCl buffer, pH 8.4, I0 ~I 0.5 M 2-mercaptoethanol, and 5 pl of 10 re.M"ferrous ammonium sulfate. From 1 to 10 ~l of the component I solution to be assayed, containing 0.1-1.0 unit of component I, is added and the volume is made up to 100/zl with distilled water. The solution is gently mixed in such a way as to avoid unnecessary aeration and immediately transferred into a piece *H. A. Barker, Biochem. Prep. 9, 25 (1962).
[51]
L-CITRA~ALATE HVDROLYASE
333
of acid-washed, soft-glass capillary tubing of 1.2-1.5 mm i.d. and 12-15 mm length. Both ends of the tubing are sealed by means of a small flame so as to leave an air space of about 1 cm at each end. The reaction mixture in the sealed capillary is then incubated in a 37 ° water bath for 90 minutes to activate the system. The activated solution in the sealed capillary is then kept at room temperature until needed for assay. Storage for several hours at room temperature does not change the activity. To obtain a sample of the activated solution, the tip of the capillary is broken off, and an aliquot is taken from the center of the liquid column by means of a 10/~l graduated Hamilton syringe. 4 The spectrophotometric assay is done in a silica cuvette (1.5 ml capacity, 10 mm light path). A 5-10 #1 aliquot of the activated enzyme solution is introduced at the bottom of a cuvette containing 1.0 ml of the Tris buffer-cysteine-FeAS assay solution at 25 °. The reaction is immediately started by addition of 25/~I of 1 M vL-citramalate by means of a small plastic delivery and stirring device. The reaction mixture is gently stirred and the absorbance is read at 250 m~ in a UV spectrophotometer, preferably provided with an automatic recording system, against a blank having about the same initial absorbance as the sample, usually 0.5 mM sodium mesaconate. The initial absolute absorbance of the reaction mixture should not exceed 2.0. The increase in absorbance at 250 m~ between 30 and 90 seconds after starting the reaction is taken as a measure of enzyme activity. The rate should be in the range of 0.02-0.4 absorbance units per minute at 25 °. A correction must be made for component I activity in the component II preparation used in the assay; this will also provide a correction for small absorbance changes resulting from nonenzymatic reactions. Units. A unit of component I activity causes an increase of 2.26 absorbance units per minute at 250 m~, corresponding to the formation of 1 micromole of mesaconate per minute, under the conditions of the assay. Specific activity is defined as units per milligram of protein determined by the method of Lowry et al., 5 using crystalline bovine serum albumin as a standard. Purification Procedure 2 All operations are carried out at 0-4 ° unless otherwise indicated. 8tep 1. Preparation o / t h e Extracts. Freshly harvested or previously frozen and thawed cell paste of Clostridium tetanomorphum strain H1, grown on a glutamate-yeast extract medium, is used as a source of Hamilton Co., Whittier, California. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
334
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
enzyme. To 500 g of cell paste is added 525 ml of 50 mM Tris-HCl buffer, pH 7.5, and the suspension is sonicated in 60 ml portions in a 10 kc Raytheon sonicator for 15 minutes. The sonicate is centrifuged for 30 minutes at 10,000 g and then for 2 hours at 55,000 g. The precipitates are resuspended in 400 ml of the same buffer and again centrifuged for 2 hours at 55,000 g. The supernatant solutions are combined and the precipitate is discarded. The pH of the extract is about 7.0. Step 2. Protamine Treatment. The protein concentration of tile extract is adjusted to 30 mg/ml with distilled water to give a final volume of 1670 ml. To this solution 0.6 volume (1 liter) of 1% protamine sulfate (salmine, Lilly) solution is added slowly with stirring during about 40 minutes. After stirring for an additional 30 minutes, the solution is centrifuged at 10,000 g for 30 minutes. The precipitate is discarded. Step 3. Ammonium Sul]ate Precipitation. Powdered ammonium sulfate (451 g) is slowly added to the enzyme (2560 ml; 10.2 mg protein per ml) with stirring to give a 30% saturated solution. After stirring for an additional 30 minutes, the precipitate is removed by centrifugation at 10,000 g for 20 minutes. An additional 1221 g of ammonium sulfate is added to bring the supernatant solution (2700 ml) to 90% saturation. After an additional 30 minutes of stirring, the solution is centrifuged at 10,000 g for 1 hour. The supernatant solution is discarded, and the precipitated protein is dissolved in 300 ml of 60 mM Tris-HC1 buffer, pH 9.0. Step 4. Heat Treatment at 55 °. The concentrated enzyme solution (498 ml; 49 mg protein per ml) is transferred to a 500 ml volumetric flask containing a magnetic stirring bar, and 1.8 ml of 14.1M 2-mercaptoethanol and 0.50 ml of 0.10M ferrous ammonium sulfate are added to give final concentrations of 50 mM and 0.1 mM respectively. The air above the solution is displaced by O~-free argon, and the flask is closed with a rubber stopper holding a thermometer. The flask is first incubated in a 37 ° water bath for 2 hours to reduce the enzyme and is then transferred to a 55 ° water bath. When the solution is stirred vigorously, it reaches a temperature of 55 ° in about 5 minutes. The flask is kept at this temperature for another 5 minutes and then is rapidly cooled in an ice bath with stirring. The denatured protein is removed by centrifugation at 10,000 g for 30 minutes and discarded. The supernatant solution is dialyzed in 1 inch diameter cellophane tubing for 24 hours against 5 liters of 0.10M Tris-HC1 buffer, pH 7.0; the buffer is changed twice. If a precipitate forms, it is removed by centrifugation. Step 5. DEAE-Sephadex ~ Column Chromatography. The dialyzed en*DEAE-cellulose may also be used to obtain about the sa.me degree of purification and yield by a slightly modified procedure (see footnote I).
[51]
L-CITRAMALATE HYDROLYASE
335
zyme solution (440 ml; 33.6 mg protein per ml) is passed into a DEAESephadex A-50 column (5.0 cm X 30 cm), previously equilibrated with 0.10M Tris-HC1 buffer, pH 7.0. Elution of the column is started witb 2800 ml of the same buffer and continued with 2 liters 0.10M TrisHC1 buffer, pH 7.0, containing 0.10M K2SO~. Fractions (25 ml) are collected at a rate of 2 ml per minute. Component I, virtually uncontaminated with component II, elutes with the Tris buffer (fractions 2890). Component II, heavily contaminated with component I activity, is eluted by the Tris-K2SO4 solution (fractions 165-190) (see Purification of Component II). Step 6. Ammonium Sul]ate Precipitation. The component I fractions from the previous step (1540 ml; 1.9 mg protein per ml) are brought to 0.83 saturation with ammonium sulfate by slow addition of 910 g of the powdered salt while the solution is stirred continuously. After stirring for another 30 minutes, the precipitated protein is separated by centrifugation at 10,000 g for 1 hour and dissolved in 50 ml of 20 mM Tris-HC1 buffer, pH 7.0. The protein solution (82 ml; 29.5 mg protein per ml) is dialyzed against 2 liters of 20 mM Tris-HC1 buffer, pH 7.0, for 12 hours with two changes of the external solution. The dialyzed solution is centrifuged at 10,000 g for 15 minutes to remove a small precipitate. Step 7. DEAE-Sephadex Column Chromatography. The dialyzed solution (100 ml; 21.5 mg protein per ml, pH 7.0) is applied to a 2.5 cm X 40 cm DEAE-Sephadex A-50 column, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7.0. The column is eluted with 3 liters of the same buffer. Fractions (25 ml) are collected at a rate of 1.0 ml per minute. Component I elutes in an almost symmetrical activity peak between fractions 73 and 96. These fractions are pooled. Step 8. Brushite Column Chromatography. The component I fractions from the previous step (564 ml; 0.26 mg protein per ml) are applied to a 1.0 cm X 10 cm brushite ~ column previously equilibrated with 20 mM Tris-HCl buffer, pH 7.0. The column is then washed with 100 ml of the same buffer. The eluate, which contains no component I activity, is collected in 25 ml fractions at the rate of 0.33 ml per minute. Component I is then eluted with 120 ml of 0.10M ammonium sulfate. Fractions (2 ml) are collected at the same rate. The activity elutes in fractions 10-24 of the second eluent. These fractions (30 ml; 1.50 mg protein per ml) are combined and carefully adjusted to pH 5.6 with 1 N HCl. The enzyme solution is then dialyzed for 18 hours against 2 liters of 15 mM sodium citrate buffer, pH 5.60; the external solution is changed twice. Step 9. CM-Cellulose Column Chromatography. The dialyzed solution ~W. T. Jenkins, BiocAem. Prep. 9, 83 (1962).
336
R E A C T I O NLEADING S TO AND FROM THE CYCLE
[51]
(35 ml; 1.29 mg protein per ml), having essentially the same pH and electrical conductivity as 15 mM sodium citrate, pH 5.6, is passed into a 1.0 cm X 10 cm CM-cellulose column previously equilibrated with the same buffer. Elution of the column is started with 50 ml of this buffer and continued with a linear salt gradient made with 50 ml of 15 mM sodium citrate, pH 5.6, in the mixing chamber and 50 ml of 0.10 M sodium citrate buffer, pH 6.5, in the reservoir. Fractions (2 ml) are collected at the rate of 0.4 ml per minute. Component I activity elutes in a narrow peak (fractions 46-50) soon after the salt gradient is started. The pooled component I fractions (10 ml; 0.95 mg protein per ml) are concentrated by precipitation with 0.80 saturated ammonium sulfate, and then dialyzed against 1 liter of 0.10M Tris-HCl buffer, pH 8.4 for 18 hours, with one change of buffer. Typical data on component I purification are given in Table I. The overall yield is 18-24% with a purification factor of about 1000. Properties of Component 12
Homogeneity and Molecular Weight. The best preparations contain more than one protein. Disc eleetrophoresis in polyacrylamide gel (anionic system) shows the presence of two major protein components of similar mobility in about equal amounts and one or more minor components. Two proteins are also seen in the Ouchterlony test 8 when highly purified component I is tested against rabbit anti-component I serum. It is not known whether only one or both proteins possess component I activity. All component I preparations after step 5 are free of component II, fl-methylaspartase 9 (threo-3-methyl-L-aspartate ammonia-lyase) and citramalate pyruvate lyase, l° The molecular weight of component I is estimated to be 46,000 ± 5000 by gel filtration using columns of Sephadex G-100 and G-200.11 Stability. Component I is best stored at a protein concentration above 1 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.4, at --10 ° or lower; the activity is stable for months under these conditions. Freezing and thawing does not significantly decrease the activity. The enzyme is quite stable from pH 7 to 11. Below pH 6 it becomes increasingly labile as the pH falls, but even at pH. 2.0 (75 mM sodium citrate buffer) 64% of the activity remains after incubation at 25 ° for 24 hours. Component I activity is considerably less stable at low than at high ionic strengths. sO. Ouchterlony, Acta Palhol. Microbiol. Scand. 26, 507 (1949). H. A. Barker, R. D. 8myth, R. M. Wilson, and H. Weissbach, J. Biol. Chem. 234, 320 (1959). ioH. A. Barker, this volume [52]. u j . R. Whitaker, Anal. Chem. 35, 1950 (1963).
[51]
L-cITrtAMXLXT~ HYDROLYASE
~
337
:E x
.~'~ l
Z
tt~
e,
<
~q
338
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
For prolonged storage, the concentration of Tris-HC1 buffer should be 0.1 M or higher. Ammonium sulfate and possible other salts can replace part of the buffer, when desirable. The enzyme is relatively stable at 37 ° or below, but loses up to 35~ of its activity when heated at 55 ° for 10 minutes under otherwise favorable conditions. Oxygen does not affect the stability of component I. Absorption Spectrum. Component I shows a typical protein absorption spectrum with Amax at 280 mt~ and Ami, at 252 mt~. The A..so,,~:A26o~,v ratio is 1.75 for a highly purified preparation (11,800 units per milligram of protein). The absorbance of a neutral 0.1% component I solution is 0.90 at 280 m~. Hydrolyase Component II Assay for Component II 1,2
This is done by the two-step procedure described in the Assay for component I, except that an excess of component I and a limiting amount of component II is used. In carrying out the activation procedure on a I00 ~l scale, I0 ~I of component I solution (for component II assay) is added to 80 /~l of Tris-mercaptoethanoI-ferrous ammonium sulfate solution. From I to 40/~l of the component II solution to be assayed, containing 0.1-1.0 unit, is added, and the volume is made up to 100 #l with water. The remainder of the procedure is identical with that used for component I assay. The activity unit and the specific activity of component II are defined as for component I. Purification Procedure 2
Steps 1 through 5 are the same as in the purification of component I. Step 6. Ammonium Sul]ate Precipitation. The component II fractions from step 5 (655 ml; 11.1 mg protein per ml) are brought to 0.60 saturation by addition of 255 g of powdered ammonium sulfate. The precipitate, collected by centrifugation at 10,000 g for 30 minutes, is dissolved in 100 ml of 20 mM Tris-HCl buffer pH 7.0. The solution is dialyzed against 2 liters of the same buffer for 12 hours with two changes of buffer. Step 7. Brushite Column Chromatography. The dialyzed solution (225 ml; 22.2 mg protein per ml) is passed into a brushite column (4.0 cm X 39 cm) previously equilibrated with 20 mM Tris-HC1 buffer pH 7.0 and is followed by 1 liter of the same buffer. The eluate is collected in 12 ml fractions at a rate of 1 ml per minute. About 75% of the added component II activity and 33% of the component I activity elute together in fractions 53-79. These fractions are combined and used for
[51]
L-CITRAMALATE HYDROLYASE
339
further purification. This step does not significantly increase the specific activity of component II but removes much of the contaminating component I. Step 8. CM-Sephadex Column Chromatography. The component II solution (332 ml; 10 mg protein per ml) is acidified to pH 5.4 with 1 N HC1 and a small precipitate is removed by centrifugation. The supernatant solution is diluted (to 500 ml) with distilled water until its conductivity is equal to that of 10 mM sodium citrate buffer pH 5.4. The solution (4.7 mg protein per ml; 126 units/rag) is then passed into a CMSephadex C-50 column (2.5 cm }( 40 era), previously equilibrated with 10 mM sodium citrate buffer pH 5.4. The column is eluted first with 250 ml of the same buffer and then with a linear salt gradient made with 500 ml of 10 mM sodium citrate pH 5.4 in the mixing vessel and 500 ml of 0.10 M sodium citrate pH 5.4 in the reservoir. Fractions (10 ml) are collected at the rate of 1 ml per minute. Component II activity elutes in two well separated peaks. The first peak (A) comes off in the passthrough fractions and the early fractions of dilute buffer (fractions 756); this peak contains about 39% of the added component II activity and is virtually free of component I activity. Peak A is used for the further described purification. The second component II peak (B) elutes in fractions 109-140 in the salt gradient; this peak contains about 49% of the added component II, but is contaminated with considerable component I activity. The peak B fraction may be put separately through steps 9 and 10 to give a final product of somewhat lower specific activity. Step 9. Ammonium Sulfate Precipitation. Fraction A (504 ml; 1.92 mg protein per ml) is brought to 0.50 saturation 1~ by addition of 158 g of ammonium sulfate. The precipitate is collected by centrifugation, dissolved in 20 ml of 0.10M Tris-HC1 buffer, pH 9.0, and the solution is centrifuged briefly to remove insoluble material. Step 10. Sephadex G-200 Column Chromatography. The enzyme solution (25 ml; 19 mg protein per ml) is passed into a 3.0 cm X 100 cm Sephadex G-200 column previously equilibrated with 0.10M TrisHC1 buffer pH 9.0. The column is eluted with the same buffer at a rate of 0.20 ml per minute; the effluent is collected in 5.0 ml fractions. Component II activity elutes in a single, ahnost symmetrical peak (fractions 64-83) located between two prominent absorbance (280 m~) peaks (fractions 47-68 and 80-110). Component II is purified about 9-fold in this step with a yield of about 83%. The active fractions (100 ml; 0.48 mg protein per ml) are concentrated by precipitation with 0.80 1.~When this step is applied to fraction B of step 8, 0.30 saturated ammonium sulfate is used.
340
R~ACTIONS
L E A D I N G TO A N D FROM T H E CYCLE
[51]
~v
Z O ~v
r~
O O
~ ~ ~i ~
o.~,~ ~ ~.~
[51]
L-CITRAMALATEHYDROLYASE
341
saturated ammonium sulfate and then are dialyzed against 1 liter of 0.10 mM Tris-HCl buffer pH 8.0 for 8 hours, with one change of buffer. Typical data on component II purification are given in Table II. The overall yield is 14~o with a purification of about 150. An additional 19% yield with a purification of about 90 (104,000 units; 900 units/rag) can be obtained by separately carrying the B fractions from step 8 through steps 9 and 10. This product is also essentially free of component I activity. It should be noted that the A and B fractions show the same difference in elution pattern when they are rechromatographed on CMSephadex columns as in the original separation. Properties of Component II
Homogeneity and Molecular Weight. Polyacrylamide gel disc electrophoresis (anionic system) shows that a quarter or a third of the protein in the best preparation (1600 units per mg protein) is one component; several other components are also present. It is not known whether component II activity is associated with the major protein component. Heterogeneity of the preparations is also indicated by the absence of correlation between activity and 280 m/~ absorbance in the elution pattern obtained by gel filtration on the Sephadex G-200 column used in the last step of component II purification. The A and B preparations of component II show a reproducible difference in their elution patterns from CM-Sephadex (see above). Apparently they contain distinct species of component II carrying different charges. The best component II preparations show no component I, fl-methylaspartase, or citramalate pyruvate lyase activity. The absence of component I protein from component II preparations is indicated by a negative reaction with anti-compound I serum (rabbit) in the Ouchterlony test. The molecular weight of component II, estimated by gel filtration through a Sephadex G-200 column equilibrated with 0.10M Tris-HC1 buffer, pH 9.0, is ~10,000 ± 20,000. This material appears to contain 8 subunits, since gel filtration at pH 7.5 shows the presence of three additional active protein species having molecular weights of about 37,000, 70,000, and 164,000. When the pH is again raised to 9.0, these lower molecular weight species reassociate to form active component II of the original molecular weight. Stability. Component II is best stored at --10 ° or lower in 0.10 M Tris-HC1 buffer pH 8.0 at a protein concentration above 2 mg/ml. Under these conditions the activity does not decline appreciably during storage for 2 months at --10% At --196 °, the activity remains constant
342
REACTIONS LEADING TO AND FROM TtIE CYCLE
[51]
for at least 8 months. Since freezing and thawing causes a 10-153 loss in activity, the enzyme should be frozen and stored in small aliquots. Component II is stable for 5 minutes at 55 ° in the presence of mercaptoethanol and ferrous ion. At 37 °, the activity is stable for at least 2 days when mercaptoethanol, ferrous ion, and component I are present. Omission of either component I or ferrous ion, greatly decreases the stability (50% loss of activity in 3 hours at 37°), whereas the further omission of mercal)tocthanol again increases tile stability (no loss of activity after 3 hours at 37°). Component II is most stable at pH 7 to 8; about 80% of the activity remains after storage for 48 hours at 4 °. Above pH 9 and below pH 6 the stability falls off rather steeply. The stability of component II at 4 ° is increased by raising the buffer or salt concentration; 0.1-0.5 M is favorable. Component II should not be frozen in the presence of mercaptoethanol. Absorption Spectrum. The absorption spectrum of component II is similar to that of component I, except that the A2som~t:Az6om~ratio is somewhat lower (1.50), probably indicative of the lesser degree of purity. The absorbance of a neutral 0.1% solution of the best preparation of component II (1600 units per mg protein) is 0.95 at 280 m~. Activators and Inhibitors. Component II is reversibly inactivated by oxygen and, in the oxidized form, is activated by incubation with fl-mercaptoethanol and some other sulfhydryl compounds; 30 mM mercaptoethanol allows maximal activation in 90 minutes at 37°; 10 mM gives about half-maximal activation under the same conditions. Ferrous ion is required for activation. Half-maximal and maximal activation require about 20 ~M and 70 #M ferrous ammonium sulfate, respectively; simple Michaelis-Menten kinetics are not observed. Ferrous ion cannot be replaced by Mg *+, Mn ++, Ca +* or Co ++. Manganese ion is somewhat inhibitory; 5 mM MnCl~_ causes about 50% inhibition in the presence of 0.5 mM ferrous ammonium sulfate. Properties of the Complete Citramalate Hydrolyase System s
Interaction o] the Components. Neither component alone possesses hydrolyase activity. To obtain maximal activity in the assay the two components must previously be incubated together under the conditions for activation described in the assays. The observed activity depends in a complex manner on several parameters. Without going into detail, these relations may be briefly summarized. The activity of each component depends upon the concentration of the other component. Activity increases with the concentration of the second
[51]
L-CITR&MALATE tIYDROLYASE
343
component until saturation is reached; this occurs when the activity of the second component is about 4 times that of the rate-limiting component. The absolute concentration of the saturating component should be about 40 units/ml. The interaction of the two components is most rapid and complete in an oxygen-free solution containing 50-200 mM Trisor ethanolamine-chloride buffer, pH 8-10, 30-50 mM mercaptoethanol, and 0.1-0.5 mM ferrous ammonium sulfate. The temperature of the solution must be above 20 ° to achieve maximal activity. At 37 °, full activation takes 60-90 minutes when the component II preparation has not previously been reduced; when it has been reduced, activation takes 15-20 minutes. Substrate Specificity. The known substrates for citramalate hydrolyase are L-(+)-citramalate, 13 L-malate, mesaconate, and fumarate. At pH 8.2 and 25 °, the K~ values for these substrates are 2.4, 6.7, 0.59, and 0.95 raM, and the relative Vmax values are 100, 256, 407, and 650, respectively, when component II is rate limiting. The same K~ values are obtained under otherwise identical conditions when component I is rate-limiting. The D-is0mers of citramalate and malate are not substrates for the enzyme. D-Malate, succinate, and L-tartrate are competitive inhibitors with K~ values of 3.5, 6.9, and 13.8 raM, respectively, determined with L-citramalate as substrate. Influence of pH on Rate. In the assay solution, containing 2.0 mM cysteine, 0.2 mM ferrous ammonium sulfate, and 60 mM Tris-HC1 buffer, maximal activity is obtained at pH 8.0-8.5; the activity is lower in more acid or alkaline solution, reaching 50% of the maximal value at about pH 7.1 and 9.3. When 2.0 mM 2-mercaptoethanol is substituted for cysteine, under otherwise identical conditions, the pH optimum is shifted to pH 6.5-7.0, and the absolute rate is decreased about 20%. Under these conditions, a half-maximal rate is observed at pH 5.7 and 7.7. It should be noted that the stability of the activity in the assay solution is greatly diminished when mercaptoethanol is substituted for cysteine; consequently with mercaptoethanol the rate of reaction decreases with time. Influence of Sulfhydryl Compounds. In the assay solution at pH 8.28.4, D-cysteine, 2-mercaptoethylamine, and DL-homocysteine are as effective as L-cysteine; 2,3-dimercapto-l-propanol is about half as effective; and mercaptoethanol, reduced glutathione, sodium thioglycolate, and 3-mercaptopropionate are ineffective. The optimal concentration of L-cysteine is 0.5-2.0 mM; at higher and lower concentrations the activity is significantly decreased. ,sp. A. von der Miihll, G. Settimj, H. Weber, and D. Arigoni, Chimia 19, 595 (1965).
344
REACTIONS LEADING TO AND FROM THE CYCLE
[52]
Divalent Cation Requirement. Ferrous ion is essential in the assay solution to stabilize the activity, even though the system has been activated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9 mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++, Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid decline of activity during the assay. Manganese ion causes the most rapid and extensive inhibition. In the presence of 0.2 mM Fe+*, the same concentration of MnCl, causes a 2 0 ~ inhibition. Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesaconate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is not available.
[52] Citramalate Pyruvate Lyase 1
By H. A. BARKER COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH Assay Method
Principle. The lyase activity can be measured most conveniently by coupling the above reaction with the reduction of pyruvate by N A D H in presence of excess lactate dehydrogenase. The decrease in absorbance at 340 ms is measured. Reagents Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M NADH, 1.0 mM MgC12, 10 mM Potassium phosphate buffer, 0.5 M, pH 7.4 Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter I The enzyme has also been called citramalase2 * If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II, p. 151. Academic Press, N e w York, 1961. *H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962). 'H. A. Barker, Biochem. Prep. 9, 25 (1962). 'A. Kornberg, Vol. I, p. 441.
L-CITRA.MALATE HYDROLYASE
331
[ 51 ] L- C i t r a m a l a t e H y d r o l y a s e
By C. C. WANQ and H. A. BARKER
coo-
I HO--C--CH3 L
CI~ _ COO L- (+) - C i t r a m a l a t e
-OOC~c/CH3 ~.
II
+
H20
H/C~.coo_ Mesaconate
A s s a y M e t h o d 1, 2
Principle. Citramalate hydrolyase activity is estimated by measuring the rate of absorbance increase at 250 n ~ resulting from the conversion of citramalate to mesaconate. The enzyme from Clostridium tetanomorphum consists of two separable protein components (I and II). The assay for each component is done by adding an excess of the other component. Maximal activity of the rate-limiting component in the assay is attained only after the two components have been incubated together ("activated") under carefully controlled conditions. Under suitable conditions the rate of absorbance increase is proportional to the concentration of the rate-limiting protein component.
Reagents Tris-HCI buffers, pH 8.2, 0.5 M and pH 8.4, 0.5 M 2-Mercaptoethanol (MET), 0.5 M. Prepare weekly Ferrous ammonium sulfate (FeAS), 10 mM. Prepare daily Tris buffer-cysteine-FeAS assay solution. This solution contains 60 mM Tris-HCl, pH 8.2, 2.0 mM L-cysteine hydrochloride, 2.0 mM NaOH, and 0.2 mM FeAS. It is made up in a graduate cylinder or similar vessel with a small air-surface to volume ratio and is mixed carefully to avoid excessive oxidation of cysteine. The temperature is adjusted to 25 °. The solution is allowed to stand at least 15 minutes; during this time the initial purple color fades (except at the surface) to give an almost color1A. It. Blair and It. A. Barker, J. Biol. Chem. 241, 400 (1966). 2C. C. Wang, Doctoral Dissertation, University of California, Berkeley, California,
1966.
332
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
less solution. The solution is stable for at least 4 hours when excessive contact with air is avoided DL-Citramalate, s disodium salt, 1.0 M, pH 7.0 Hydrolyase component I solution (for component II assay), 400 units per milliliter of 20 mM Tris-HC1 buffer, pH 7.0. The preparation should have a specific activity of at least 300 units per milligram of protein and should be free of component II activity Hydrolyase component I solution (for component I assay). The solution, diluted when necessary in 20 mM Tris-HC1 buffer, pH 7.0, should contain between 10 and 1000 units/ml Hydrolyase component II solution (for component I assay), 400 units per milliliter of 20 mM Tris-HCl buffer, pH 8.0, and 50 mM K2S04. The preparation should have a specific activity of at least 100 units per milligram of protein and should be relatively free of component I activity (less than 5 ~ of the component II specific activity) Hydrolyase component II solution (for component II assay). The solution, diluted when necessary in 20 mM Tris-HCl buffer, pH 8.0, and 50 mM K2S0~, should contain between 10 and 1000 units/ml Hydrolyase Component I Assay for Component I Procedure. The assay involves two distinct steps: (a) the "activation" of the system by incubating a rate limiting amount of component I with an excess of component II under specific conditions, and (b) the spectrophotometric assay proper, using an aliquot of the activated enzyme solution. The activation of the system is conveniently done in a reaction mixture having a volume of 100 pl, although a smaller volume may be used. On the I00 pl scale, 10 pl of hydrolyase component II solution (for component I assay) is added to 80 ~l of a freshly prepared solution (in a 0.5 ml test tube at 0 °) containing 10 pl 0.5M Tris-HCl buffer, pH 8.4, I0 ~I 0.5 M 2-mercaptoethanol, and 5 pl of 10 re.M"ferrous ammonium sulfate. From 1 to 10 ~l of the component I solution to be assayed, containing 0.1-1.0 unit of component I, is added and the volume is made up to 100/zl with distilled water. The solution is gently mixed in such a way as to avoid unnecessary aeration and immediately transferred into a piece *H. A. Barker, Biochem. Prep. 9, 25 (1962).
[51]
L-CITRA~ALATE HVDROLYASE
333
of acid-washed, soft-glass capillary tubing of 1.2-1.5 mm i.d. and 12-15 mm length. Both ends of the tubing are sealed by means of a small flame so as to leave an air space of about 1 cm at each end. The reaction mixture in the sealed capillary is then incubated in a 37 ° water bath for 90 minutes to activate the system. The activated solution in the sealed capillary is then kept at room temperature until needed for assay. Storage for several hours at room temperature does not change the activity. To obtain a sample of the activated solution, the tip of the capillary is broken off, and an aliquot is taken from the center of the liquid column by means of a 10/~l graduated Hamilton syringe. 4 The spectrophotometric assay is done in a silica cuvette (1.5 ml capacity, 10 mm light path). A 5-10 #1 aliquot of the activated enzyme solution is introduced at the bottom of a cuvette containing 1.0 ml of the Tris buffer-cysteine-FeAS assay solution at 25 °. The reaction is immediately started by addition of 25/~I of 1 M vL-citramalate by means of a small plastic delivery and stirring device. The reaction mixture is gently stirred and the absorbance is read at 250 m~ in a UV spectrophotometer, preferably provided with an automatic recording system, against a blank having about the same initial absorbance as the sample, usually 0.5 mM sodium mesaconate. The initial absolute absorbance of the reaction mixture should not exceed 2.0. The increase in absorbance at 250 m~ between 30 and 90 seconds after starting the reaction is taken as a measure of enzyme activity. The rate should be in the range of 0.02-0.4 absorbance units per minute at 25 °. A correction must be made for component I activity in the component II preparation used in the assay; this will also provide a correction for small absorbance changes resulting from nonenzymatic reactions. Units. A unit of component I activity causes an increase of 2.26 absorbance units per minute at 250 m~, corresponding to the formation of 1 micromole of mesaconate per minute, under the conditions of the assay. Specific activity is defined as units per milligram of protein determined by the method of Lowry et al., 5 using crystalline bovine serum albumin as a standard. Purification Procedure 2 All operations are carried out at 0-4 ° unless otherwise indicated. 8tep 1. Preparation o / t h e Extracts. Freshly harvested or previously frozen and thawed cell paste of Clostridium tetanomorphum strain H1, grown on a glutamate-yeast extract medium, is used as a source of Hamilton Co., Whittier, California. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
334
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
enzyme. To 500 g of cell paste is added 525 ml of 50 mM Tris-HCl buffer, pH 7.5, and the suspension is sonicated in 60 ml portions in a 10 kc Raytheon sonicator for 15 minutes. The sonicate is centrifuged for 30 minutes at 10,000 g and then for 2 hours at 55,000 g. The precipitates are resuspended in 400 ml of the same buffer and again centrifuged for 2 hours at 55,000 g. The supernatant solutions are combined and the precipitate is discarded. The pH of the extract is about 7.0. Step 2. Protamine Treatment. The protein concentration of tile extract is adjusted to 30 mg/ml with distilled water to give a final volume of 1670 ml. To this solution 0.6 volume (1 liter) of 1% protamine sulfate (salmine, Lilly) solution is added slowly with stirring during about 40 minutes. After stirring for an additional 30 minutes, the solution is centrifuged at 10,000 g for 30 minutes. The precipitate is discarded. Step 3. Ammonium Sul]ate Precipitation. Powdered ammonium sulfate (451 g) is slowly added to the enzyme (2560 ml; 10.2 mg protein per ml) with stirring to give a 30% saturated solution. After stirring for an additional 30 minutes, the precipitate is removed by centrifugation at 10,000 g for 20 minutes. An additional 1221 g of ammonium sulfate is added to bring the supernatant solution (2700 ml) to 90% saturation. After an additional 30 minutes of stirring, the solution is centrifuged at 10,000 g for 1 hour. The supernatant solution is discarded, and the precipitated protein is dissolved in 300 ml of 60 mM Tris-HC1 buffer, pH 9.0. Step 4. Heat Treatment at 55 °. The concentrated enzyme solution (498 ml; 49 mg protein per ml) is transferred to a 500 ml volumetric flask containing a magnetic stirring bar, and 1.8 ml of 14.1M 2-mercaptoethanol and 0.50 ml of 0.10M ferrous ammonium sulfate are added to give final concentrations of 50 mM and 0.1 mM respectively. The air above the solution is displaced by O~-free argon, and the flask is closed with a rubber stopper holding a thermometer. The flask is first incubated in a 37 ° water bath for 2 hours to reduce the enzyme and is then transferred to a 55 ° water bath. When the solution is stirred vigorously, it reaches a temperature of 55 ° in about 5 minutes. The flask is kept at this temperature for another 5 minutes and then is rapidly cooled in an ice bath with stirring. The denatured protein is removed by centrifugation at 10,000 g for 30 minutes and discarded. The supernatant solution is dialyzed in 1 inch diameter cellophane tubing for 24 hours against 5 liters of 0.10M Tris-HC1 buffer, pH 7.0; the buffer is changed twice. If a precipitate forms, it is removed by centrifugation. Step 5. DEAE-Sephadex ~ Column Chromatography. The dialyzed en*DEAE-cellulose may also be used to obtain about the sa.me degree of purification and yield by a slightly modified procedure (see footnote I).
[51]
L-CITRAMALATE HYDROLYASE
335
zyme solution (440 ml; 33.6 mg protein per ml) is passed into a DEAESephadex A-50 column (5.0 cm X 30 cm), previously equilibrated with 0.10M Tris-HC1 buffer, pH 7.0. Elution of the column is started witb 2800 ml of the same buffer and continued with 2 liters 0.10M TrisHC1 buffer, pH 7.0, containing 0.10M K2SO~. Fractions (25 ml) are collected at a rate of 2 ml per minute. Component I, virtually uncontaminated with component II, elutes with the Tris buffer (fractions 2890). Component II, heavily contaminated with component I activity, is eluted by the Tris-K2SO4 solution (fractions 165-190) (see Purification of Component II). Step 6. Ammonium Sul]ate Precipitation. The component I fractions from the previous step (1540 ml; 1.9 mg protein per ml) are brought to 0.83 saturation with ammonium sulfate by slow addition of 910 g of the powdered salt while the solution is stirred continuously. After stirring for another 30 minutes, the precipitated protein is separated by centrifugation at 10,000 g for 1 hour and dissolved in 50 ml of 20 mM Tris-HC1 buffer, pH 7.0. The protein solution (82 ml; 29.5 mg protein per ml) is dialyzed against 2 liters of 20 mM Tris-HC1 buffer, pH 7.0, for 12 hours with two changes of the external solution. The dialyzed solution is centrifuged at 10,000 g for 15 minutes to remove a small precipitate. Step 7. DEAE-Sephadex Column Chromatography. The dialyzed solution (100 ml; 21.5 mg protein per ml, pH 7.0) is applied to a 2.5 cm X 40 cm DEAE-Sephadex A-50 column, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7.0. The column is eluted with 3 liters of the same buffer. Fractions (25 ml) are collected at a rate of 1.0 ml per minute. Component I elutes in an almost symmetrical activity peak between fractions 73 and 96. These fractions are pooled. Step 8. Brushite Column Chromatography. The component I fractions from the previous step (564 ml; 0.26 mg protein per ml) are applied to a 1.0 cm X 10 cm brushite ~ column previously equilibrated with 20 mM Tris-HCl buffer, pH 7.0. The column is then washed with 100 ml of the same buffer. The eluate, which contains no component I activity, is collected in 25 ml fractions at the rate of 0.33 ml per minute. Component I is then eluted with 120 ml of 0.10M ammonium sulfate. Fractions (2 ml) are collected at the same rate. The activity elutes in fractions 10-24 of the second eluent. These fractions (30 ml; 1.50 mg protein per ml) are combined and carefully adjusted to pH 5.6 with 1 N HCl. The enzyme solution is then dialyzed for 18 hours against 2 liters of 15 mM sodium citrate buffer, pH 5.60; the external solution is changed twice. Step 9. CM-Cellulose Column Chromatography. The dialyzed solution ~W. T. Jenkins, BiocAem. Prep. 9, 83 (1962).
336
R E A C T I O NLEADING S TO AND FROM THE CYCLE
[51]
(35 ml; 1.29 mg protein per ml), having essentially the same pH and electrical conductivity as 15 mM sodium citrate, pH 5.6, is passed into a 1.0 cm X 10 cm CM-cellulose column previously equilibrated with the same buffer. Elution of the column is started with 50 ml of this buffer and continued with a linear salt gradient made with 50 ml of 15 mM sodium citrate, pH 5.6, in the mixing chamber and 50 ml of 0.10 M sodium citrate buffer, pH 6.5, in the reservoir. Fractions (2 ml) are collected at the rate of 0.4 ml per minute. Component I activity elutes in a narrow peak (fractions 46-50) soon after the salt gradient is started. The pooled component I fractions (10 ml; 0.95 mg protein per ml) are concentrated by precipitation with 0.80 saturated ammonium sulfate, and then dialyzed against 1 liter of 0.10M Tris-HCl buffer, pH 8.4 for 18 hours, with one change of buffer. Typical data on component I purification are given in Table I. The overall yield is 18-24% with a purification factor of about 1000. Properties of Component 12
Homogeneity and Molecular Weight. The best preparations contain more than one protein. Disc eleetrophoresis in polyacrylamide gel (anionic system) shows the presence of two major protein components of similar mobility in about equal amounts and one or more minor components. Two proteins are also seen in the Ouchterlony test 8 when highly purified component I is tested against rabbit anti-component I serum. It is not known whether only one or both proteins possess component I activity. All component I preparations after step 5 are free of component II, fl-methylaspartase 9 (threo-3-methyl-L-aspartate ammonia-lyase) and citramalate pyruvate lyase, l° The molecular weight of component I is estimated to be 46,000 ± 5000 by gel filtration using columns of Sephadex G-100 and G-200.11 Stability. Component I is best stored at a protein concentration above 1 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.4, at --10 ° or lower; the activity is stable for months under these conditions. Freezing and thawing does not significantly decrease the activity. The enzyme is quite stable from pH 7 to 11. Below pH 6 it becomes increasingly labile as the pH falls, but even at pH. 2.0 (75 mM sodium citrate buffer) 64% of the activity remains after incubation at 25 ° for 24 hours. Component I activity is considerably less stable at low than at high ionic strengths. sO. Ouchterlony, Acta Palhol. Microbiol. Scand. 26, 507 (1949). H. A. Barker, R. D. 8myth, R. M. Wilson, and H. Weissbach, J. Biol. Chem. 234, 320 (1959). ioH. A. Barker, this volume [52]. u j . R. Whitaker, Anal. Chem. 35, 1950 (1963).
[51]
L-cITrtAMXLXT~ HYDROLYASE
~
337
:E x
.~'~ l
Z
tt~
e,
<
~q
338
REACTIONS LEADING TO AND FROM THE CYCLE
[51]
For prolonged storage, the concentration of Tris-HC1 buffer should be 0.1 M or higher. Ammonium sulfate and possible other salts can replace part of the buffer, when desirable. The enzyme is relatively stable at 37 ° or below, but loses up to 35~ of its activity when heated at 55 ° for 10 minutes under otherwise favorable conditions. Oxygen does not affect the stability of component I. Absorption Spectrum. Component I shows a typical protein absorption spectrum with Amax at 280 mt~ and Ami, at 252 mt~. The A..so,,~:A26o~,v ratio is 1.75 for a highly purified preparation (11,800 units per milligram of protein). The absorbance of a neutral 0.1% component I solution is 0.90 at 280 m~. Hydrolyase Component II Assay for Component II 1,2
This is done by the two-step procedure described in the Assay for component I, except that an excess of component I and a limiting amount of component II is used. In carrying out the activation procedure on a I00 ~l scale, I0 ~I of component I solution (for component II assay) is added to 80 /~l of Tris-mercaptoethanoI-ferrous ammonium sulfate solution. From I to 40/~l of the component II solution to be assayed, containing 0.1-1.0 unit, is added, and the volume is made up to 100 #l with water. The remainder of the procedure is identical with that used for component I assay. The activity unit and the specific activity of component II are defined as for component I. Purification Procedure 2
Steps 1 through 5 are the same as in the purification of component I. Step 6. Ammonium Sul]ate Precipitation. The component II fractions from step 5 (655 ml; 11.1 mg protein per ml) are brought to 0.60 saturation by addition of 255 g of powdered ammonium sulfate. The precipitate, collected by centrifugation at 10,000 g for 30 minutes, is dissolved in 100 ml of 20 mM Tris-HCl buffer pH 7.0. The solution is dialyzed against 2 liters of the same buffer for 12 hours with two changes of buffer. Step 7. Brushite Column Chromatography. The dialyzed solution (225 ml; 22.2 mg protein per ml) is passed into a brushite column (4.0 cm X 39 cm) previously equilibrated with 20 mM Tris-HC1 buffer pH 7.0 and is followed by 1 liter of the same buffer. The eluate is collected in 12 ml fractions at a rate of 1 ml per minute. About 75% of the added component II activity and 33% of the component I activity elute together in fractions 53-79. These fractions are combined and used for
[51]
L-CITRAMALATE HYDROLYASE
339
further purification. This step does not significantly increase the specific activity of component II but removes much of the contaminating component I. Step 8. CM-Sephadex Column Chromatography. The component II solution (332 ml; 10 mg protein per ml) is acidified to pH 5.4 with 1 N HC1 and a small precipitate is removed by centrifugation. The supernatant solution is diluted (to 500 ml) with distilled water until its conductivity is equal to that of 10 mM sodium citrate buffer pH 5.4. The solution (4.7 mg protein per ml; 126 units/rag) is then passed into a CMSephadex C-50 column (2.5 cm }( 40 era), previously equilibrated with 10 mM sodium citrate buffer pH 5.4. The column is eluted first with 250 ml of the same buffer and then with a linear salt gradient made with 500 ml of 10 mM sodium citrate pH 5.4 in the mixing vessel and 500 ml of 0.10 M sodium citrate pH 5.4 in the reservoir. Fractions (10 ml) are collected at the rate of 1 ml per minute. Component II activity elutes in two well separated peaks. The first peak (A) comes off in the passthrough fractions and the early fractions of dilute buffer (fractions 756); this peak contains about 39% of the added component II activity and is virtually free of component I activity. Peak A is used for the further described purification. The second component II peak (B) elutes in fractions 109-140 in the salt gradient; this peak contains about 49% of the added component II, but is contaminated with considerable component I activity. The peak B fraction may be put separately through steps 9 and 10 to give a final product of somewhat lower specific activity. Step 9. Ammonium Sulfate Precipitation. Fraction A (504 ml; 1.92 mg protein per ml) is brought to 0.50 saturation 1~ by addition of 158 g of ammonium sulfate. The precipitate is collected by centrifugation, dissolved in 20 ml of 0.10M Tris-HC1 buffer, pH 9.0, and the solution is centrifuged briefly to remove insoluble material. Step 10. Sephadex G-200 Column Chromatography. The enzyme solution (25 ml; 19 mg protein per ml) is passed into a 3.0 cm X 100 cm Sephadex G-200 column previously equilibrated with 0.10M TrisHC1 buffer pH 9.0. The column is eluted with the same buffer at a rate of 0.20 ml per minute; the effluent is collected in 5.0 ml fractions. Component II activity elutes in a single, ahnost symmetrical peak (fractions 64-83) located between two prominent absorbance (280 m~) peaks (fractions 47-68 and 80-110). Component II is purified about 9-fold in this step with a yield of about 83%. The active fractions (100 ml; 0.48 mg protein per ml) are concentrated by precipitation with 0.80 1.~When this step is applied to fraction B of step 8, 0.30 saturated ammonium sulfate is used.
340
R~ACTIONS
L E A D I N G TO A N D FROM T H E CYCLE
[51]
~v
Z O ~v
r~
O O
~ ~ ~i ~
o.~,~ ~ ~.~
[51]
L-CITRAMALATEHYDROLYASE
341
saturated ammonium sulfate and then are dialyzed against 1 liter of 0.10 mM Tris-HCl buffer pH 8.0 for 8 hours, with one change of buffer. Typical data on component II purification are given in Table II. The overall yield is 14~o with a purification of about 150. An additional 19% yield with a purification of about 90 (104,000 units; 900 units/rag) can be obtained by separately carrying the B fractions from step 8 through steps 9 and 10. This product is also essentially free of component I activity. It should be noted that the A and B fractions show the same difference in elution pattern when they are rechromatographed on CMSephadex columns as in the original separation. Properties of Component II
Homogeneity and Molecular Weight. Polyacrylamide gel disc electrophoresis (anionic system) shows that a quarter or a third of the protein in the best preparation (1600 units per mg protein) is one component; several other components are also present. It is not known whether component II activity is associated with the major protein component. Heterogeneity of the preparations is also indicated by the absence of correlation between activity and 280 m/~ absorbance in the elution pattern obtained by gel filtration on the Sephadex G-200 column used in the last step of component II purification. The A and B preparations of component II show a reproducible difference in their elution patterns from CM-Sephadex (see above). Apparently they contain distinct species of component II carrying different charges. The best component II preparations show no component I, fl-methylaspartase, or citramalate pyruvate lyase activity. The absence of component I protein from component II preparations is indicated by a negative reaction with anti-compound I serum (rabbit) in the Ouchterlony test. The molecular weight of component II, estimated by gel filtration through a Sephadex G-200 column equilibrated with 0.10M Tris-HC1 buffer, pH 9.0, is ~10,000 ± 20,000. This material appears to contain 8 subunits, since gel filtration at pH 7.5 shows the presence of three additional active protein species having molecular weights of about 37,000, 70,000, and 164,000. When the pH is again raised to 9.0, these lower molecular weight species reassociate to form active component II of the original molecular weight. Stability. Component II is best stored at --10 ° or lower in 0.10 M Tris-HC1 buffer pH 8.0 at a protein concentration above 2 mg/ml. Under these conditions the activity does not decline appreciably during storage for 2 months at --10% At --196 °, the activity remains constant
342
REACTIONS LEADING TO AND FROM TtIE CYCLE
[51]
for at least 8 months. Since freezing and thawing causes a 10-153 loss in activity, the enzyme should be frozen and stored in small aliquots. Component II is stable for 5 minutes at 55 ° in the presence of mercaptoethanol and ferrous ion. At 37 °, the activity is stable for at least 2 days when mercaptoethanol, ferrous ion, and component I are present. Omission of either component I or ferrous ion, greatly decreases the stability (50% loss of activity in 3 hours at 37°), whereas the further omission of mercal)tocthanol again increases tile stability (no loss of activity after 3 hours at 37°). Component II is most stable at pH 7 to 8; about 80% of the activity remains after storage for 48 hours at 4 °. Above pH 9 and below pH 6 the stability falls off rather steeply. The stability of component II at 4 ° is increased by raising the buffer or salt concentration; 0.1-0.5 M is favorable. Component II should not be frozen in the presence of mercaptoethanol. Absorption Spectrum. The absorption spectrum of component II is similar to that of component I, except that the A2som~t:Az6om~ratio is somewhat lower (1.50), probably indicative of the lesser degree of purity. The absorbance of a neutral 0.1% solution of the best preparation of component II (1600 units per mg protein) is 0.95 at 280 m~. Activators and Inhibitors. Component II is reversibly inactivated by oxygen and, in the oxidized form, is activated by incubation with fl-mercaptoethanol and some other sulfhydryl compounds; 30 mM mercaptoethanol allows maximal activation in 90 minutes at 37°; 10 mM gives about half-maximal activation under the same conditions. Ferrous ion is required for activation. Half-maximal and maximal activation require about 20 ~M and 70 #M ferrous ammonium sulfate, respectively; simple Michaelis-Menten kinetics are not observed. Ferrous ion cannot be replaced by Mg *+, Mn ++, Ca +* or Co ++. Manganese ion is somewhat inhibitory; 5 mM MnCl~_ causes about 50% inhibition in the presence of 0.5 mM ferrous ammonium sulfate. Properties of the Complete Citramalate Hydrolyase System s
Interaction o] the Components. Neither component alone possesses hydrolyase activity. To obtain maximal activity in the assay the two components must previously be incubated together under the conditions for activation described in the assays. The observed activity depends in a complex manner on several parameters. Without going into detail, these relations may be briefly summarized. The activity of each component depends upon the concentration of the other component. Activity increases with the concentration of the second
[51]
L-CITR&MALATE tIYDROLYASE
343
component until saturation is reached; this occurs when the activity of the second component is about 4 times that of the rate-limiting component. The absolute concentration of the saturating component should be about 40 units/ml. The interaction of the two components is most rapid and complete in an oxygen-free solution containing 50-200 mM Trisor ethanolamine-chloride buffer, pH 8-10, 30-50 mM mercaptoethanol, and 0.1-0.5 mM ferrous ammonium sulfate. The temperature of the solution must be above 20 ° to achieve maximal activity. At 37 °, full activation takes 60-90 minutes when the component II preparation has not previously been reduced; when it has been reduced, activation takes 15-20 minutes. Substrate Specificity. The known substrates for citramalate hydrolyase are L-(+)-citramalate, 13 L-malate, mesaconate, and fumarate. At pH 8.2 and 25 °, the K~ values for these substrates are 2.4, 6.7, 0.59, and 0.95 raM, and the relative Vmax values are 100, 256, 407, and 650, respectively, when component II is rate limiting. The same K~ values are obtained under otherwise identical conditions when component I is rate-limiting. The D-is0mers of citramalate and malate are not substrates for the enzyme. D-Malate, succinate, and L-tartrate are competitive inhibitors with K~ values of 3.5, 6.9, and 13.8 raM, respectively, determined with L-citramalate as substrate. Influence of pH on Rate. In the assay solution, containing 2.0 mM cysteine, 0.2 mM ferrous ammonium sulfate, and 60 mM Tris-HC1 buffer, maximal activity is obtained at pH 8.0-8.5; the activity is lower in more acid or alkaline solution, reaching 50% of the maximal value at about pH 7.1 and 9.3. When 2.0 mM 2-mercaptoethanol is substituted for cysteine, under otherwise identical conditions, the pH optimum is shifted to pH 6.5-7.0, and the absolute rate is decreased about 20%. Under these conditions, a half-maximal rate is observed at pH 5.7 and 7.7. It should be noted that the stability of the activity in the assay solution is greatly diminished when mercaptoethanol is substituted for cysteine; consequently with mercaptoethanol the rate of reaction decreases with time. Influence of Sulfhydryl Compounds. In the assay solution at pH 8.28.4, D-cysteine, 2-mercaptoethylamine, and DL-homocysteine are as effective as L-cysteine; 2,3-dimercapto-l-propanol is about half as effective; and mercaptoethanol, reduced glutathione, sodium thioglycolate, and 3-mercaptopropionate are ineffective. The optimal concentration of L-cysteine is 0.5-2.0 mM; at higher and lower concentrations the activity is significantly decreased. ,sp. A. von der Miihll, G. Settimj, H. Weber, and D. Arigoni, Chimia 19, 595 (1965).
344
REACTIONS LEADING TO AND FROM THE CYCLE
[52]
Divalent Cation Requirement. Ferrous ion is essential in the assay solution to stabilize the activity, even though the system has been activated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9 mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++, Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid decline of activity during the assay. Manganese ion causes the most rapid and extensive inhibition. In the presence of 0.2 mM Fe+*, the same concentration of MnCl, causes a 2 0 ~ inhibition. Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesaconate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is not available.
[52] Citramalate Pyruvate Lyase 1
By H. A. BARKER COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH Assay Method
Principle. The lyase activity can be measured most conveniently by coupling the above reaction with the reduction of pyruvate by N A D H in presence of excess lactate dehydrogenase. The decrease in absorbance at 340 ms is measured. Reagents Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M NADH, 1.0 mM MgC12, 10 mM Potassium phosphate buffer, 0.5 M, pH 7.4 Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter I The enzyme has also been called citramalase2 * If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II, p. 151. Academic Press, N e w York, 1961. *H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962). 'H. A. Barker, Biochem. Prep. 9, 25 (1962). 'A. Kornberg, Vol. I, p. 441.