ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Mechanism Purification THOMAS
and Properties
of Feedback
Inhibition
of a Feedback-Resistant
S. SOPER2, GEORGE Department
173, 362-374 (1976)
J. DOELLGAST3,
of Biochemistry,
Purdue Received
University, August
by Leucine c&opropylmalate
AND
GUNTER
West Lafayette,
Indiana
Synthase’
B. KOHLHAW4 47907
11, 1975
A mutationally altered, n-leucine-resistant form of a-isopropylmalate synthase, the first committed enzyme in leucine biosynthesis, has been purified to near homogeneity. Comparison of the feedback-resistant enzyme with its wild-type parent shows the following: Both enzymes are very similar with respect to substrate specificity and maximal activity, but the feedback-resistant enzyme has a greater affinity for one of the substrates, a-ketoisovalerate. The feedback-resistant enzyme is about three orders of magnitude less sensitive to L-leucine than wild-type enzyme. By contrast, it is slightly more sensitive to r,-isoleucine, the only other naturally occurring amino acid known to inhibit a-isopropylmalate synthase. Results of chemical densensitization experiments suggest that the leucine, isoleucine, and active sites are distinct. The kinetic pattern of leucine inhibition at pH 7.0 shows that leucine is a noncompetitive inhibitor with respect to both substrates with wild-type enzyme, whereas the weak inhibition by leucine of the feedback-resistant enzyme is of a competitive type. Intersubunit cross-linking of the feedback-resistant enzyme followed by gel electrophoresis in sodium dodecyl sulfate reveals the presence of monomers, dimers, and tetramers with molecular weights of approximately 52,000, 110,000, and 200,000, respectively. Very similar results had been obtained with wild-type enzyme. Sedimentation equilibrium analyses indicate that both enzymes exist as associating-dissociating systems that can be adequately described by either a monomer-tetramer or a monomer-dimer-tetramer equilibrium. With the feedback-resistant enzyme, the equilibrium constant for the monomer-tetramer equilibrium. K, = [A,]/[A14, is 1 x 10Ly M-~, compared with 9 x 10’” Mm3 for wild-type enzyme. This suggests a stronger tendency of the subunits of the feedback-resistant enzyme to aggregate, a conclusion supported by gel filtration experiments. These results, together with previous observations that wild-type enzyme is dissociated by leucine whereas the feedback-resistant enzyme is not, suggest that efficient inhibition of cY-isopropylmalate synthase by leucine may be coupled to a relatively loose arrangement of subunits within the oligomeric structure of the enzyme.
The leucine biosynthetic pathway in Salmonella typhimurium consists of three unique enzymes: cY-isopropylmalate syn-
thase (EC 4.1.3.12), isopropylmalate isomerase (EC 4.2.1.33), and /3-isopropylmalate dehydrogenase (EC 1.1.1.85). The product of these reactions, cr-ketoisocaproate, is converted to leucine by transaminase B (EC 2.6.1.6). The pathway is controlled by both end-product repression and end-product (feedback) inhibition (l-3). Feedback inhibition by leucine is directed toward the first enzyme in the pathway, which catalyzes the condensation of acetylCoA and cr-ketoisovalerate to give a-isopropylmalate and CoA. We have previ-
’ This work was supported by Research Grant GM 15102 from the National Institute of General Medical Sciences. Journal Paper No. 5992 of the Agricultural Experiment Station, Purdue University. * Present address: Rockefeller University, New York, N. Y. 10021. 3 Present address: Tufts University, School of Medicine, 136 Harrison Ave., Boston, Mass. 02111. *To whom all correspondence should be addressed. 362 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
FEEDBACK
INHIBITION
ously reported that wild-type a-isopropylmalate synthase has a subunit molecular weight of 50,000 +- 3000 (4, 51, and that, in the absence of ligands, the enzyme exists in a rapid association-dissociation equilibrium between monomers, tetramers, and possibly dimers (5). The presence of one substrate together with an analog of the other favors formation of tetramers (5) while the presence of the feedback inhibitor causes dissociation, usually leading to a predominance of dimers (2, 4, 6, 7). The stabilization of dimers by leucine is probably related to the fact that there is only one saturable leucine binding site per dimer (8). The availability of a strain producing constitutively high levels of a feedbackresistant a-isopropylmalate synthase (strain CV-241; 9) made it possible to embark on an extensive comparison of this enzyme with wild-type cY-isopropylmalate synthase and, thus, opened a new avenue for the study of leucine inhibition. It has already been established that the feedback-resistant enzyme is approximately three orders of magnitude less sensitive to leucine than its wild-type counterpart. Thus, in 80 mM potassium phosphate buffer, pH 6.8, and in the presence of 0.8 mM acetyl-CoA and 4 mM a-ketoisovalerate, the apparent K i value was found to be 20 mM for the feedback-resistant and 22 PM for wild-type enzyme (8). It has also been shown that the feedback-resistant enzyme is not dissociated by leucine, even when inhibitory concentrations of the amino acid are employed (2, 8, 10). This suggested that the mutation to feedback resistance has led to changes in the quaternary structure of the enzyme. We report in this paper that wild-type and feedback-resistant a-isopropylmalate synthases of comparable purity exhibit several subtle but important differences in addition to the large difference in leucine sensitivity. Of particular significance are results that indicate that the feedback-resistant enzyme apparently still exists in an equilibrium involving monomers, dimers, and tetramers, but that it has a greater tendency to aggregate within the framework of this equilibrium when compared with wild-type enzyme. We discuss
363
BY LEUCINE
these results in terms of an inverse relationship between the strength of subunit interactions and the extent of inhibition by leucine. EXPERIMENTAL
PROCEDURE
Materials Chemical. Special chemicals and their suppliers were: i,-leucine, L-isoleucine, n-leucine, n-isoleutine, N-methyl-n,n-leucine, n-leucine amide, and pyridoxal-5’-phosphate, Sigma; L-valine, N-methylD,L-isoleucine, a-ketoisovalerate, and 5,5’-dithiobis(2-nitrobenzoate), Calbiochem; L-isoleucine amide, Cycle; CoA (lithium salt, “chromatopure,” 90% reduced CoA), 3’-dephospho-CoA and (l,N”-ethenojCoA, PL-Biochemicals; Tris5 base, enzyme grade, Schwart=Mann; polyethyland urea, “ultrapure,” ene glycol 6000 (molecular weights, 600&7500), Matheson, Coleman and Bell. All other commercially obtained chemicals were of the best available grade. 5’,5’,5’-trifluoroleucine, azaleucine, and thiaisoleucine were gifts from H. E. Umbarger, Purdue University. A generous gift of trifluoroleucine was also obtained from H. S. Anker and D. F. Steiner, University of Chicago. Acetyl-CoA and propionyl-CoA were synthesized from CoA and the appropriate acid anhydride, following the general procedure of Simon and Shemin (11). Dimethylsuberimidate was prepared according to Davies and Stark
(12). Biological. The two strains utilized are both derivatives of Salmonella typhimurium LT-2. Strain CV-19 (genotype ara-9 gal-205 flrB), isolated as a 5’,5’,5’-trifluoroleucine-resistant mutant, served as the source for wild-type enzyme. This strain carries a mutation unlinked to the leucine operon CjlrB; 13) which results in constitutively high levels of the leucine as well as the isoleucine-valine biosynthetic enzymes (14). Strain CV-241 (genotype 1euA 2010 gal-205 flrB) served as the source of feedback-resistant enzyme. It was the result of a transduction cross (91, and carries the flrB marker and, in addition, a mutation in the structural gene for a-isopropylmalate synthase (leaA 2010), which renders this enzyme much less sensitive to inhibition by leucine (8, 14). Cells were grown and harvested as described previously (2). Methods Assay for a-isopropylmalate synthase. An endpoint assay based on the determination of free CoA with 5,5’-dithiobis-(2-nitrobenzoate) was used throughout this study. The standard assay mixture contained potassium phosphate buffer, pH 7.0, 200 s Abbreviation aminomethane.
used: Tris,
tris-(hydroxymethyl)-
364
SOPER,
DOELLGAST
mM; acetyl-CoA, 0.8 mM; a-ketoisovalerate, 4.0 mbr; and enzyme to give a final absorbance of no more than 0.3 units at 412 nm and 37°C. The assay volume was 0.125 ml. Under these conditions, the assay was linear with time for at least 10 min. The reaction was stopped by addition of 3 vol of ethanol. To this were added 2 vol of a 1 mM solution of 5,5’-dithiobis(2-nitrobenzoate) in 50 rnM Tris-HCl buffer, pH 7.5. The molar extinction coefficient of the 5-carboxy-4nitrothiophenolate ion formed is 13,600 at 412 nm (15). Specific activity of the enzyme is defined as *moles of CoA formed per h per mg of protein. Protein determination. Protein was determined by the biuret method (16) with bovine serum albumin as standard. Interference by glycerol was corrected for with appropriate standard curves. Purification of feedback-resistant a-isopropylmalate synthase. (a) Preparation of crude extract. A 1:l (w/v) suspension of cells in 0.1 M potassium phosphate buffer, pH 6.8, was passed through an Aminco French’Pressure Cell in 40-ml portions, at a pressure of 540 atm. The suspension was then diluted with 2 parts of the same buffer and centrifuged at 48,OOOgfor 1 h. The pellet was discarded. (b) Streptomycin sulfate precipitation. An amount of streptomycin sulfate equal to 25% of the total protein (by weight), applied as a 10% (w/v) solution in 0.1 M potassium phosphate, pH 6.8, was slowly added to the crude extract. The extract was then centrifuged at 28,000g for 30 min, and the pellet discarded. (c) Fractionation zuith polyethylene glycol. The protein concentration was adjusted to 16 to 20 mg/ml by addition of 0.1 M potassium phosphate buffer, pH 6.8. Granulated polyethylene glycol6000 was slowly added to give a concentration of 6% (w/v). The precipitate was collected by centrifugation for 10 min at 10,OOOg and dissolved in the aforementioned buffer to give a solution containing no less than 10 mg of protein/ml. (d) Hydroxyapatite chromatography. One milliliter of hydroxyapatite suspension (made up in 0.1 M phosphate buffer and measured when settled) per 50 mg of protein was added to the solution obtained in the previous step. The suspension was stirred for 1 h at a slow speed (sufficient to keep the hydroxyapatite in suspension) and centrifuged for 10 min at 150g. The pellet was washed three times with 0.16 M potassium phosphate buffer, pH 6.8, resuspended in the same buffer, and poured into a glass column which already contained 1 ml of settled hydroxyapatitel2 mg of protein. The added hydroxyapatite was allowed to settle, and was washed with two column volumes of 0.16 M potassium phosphate buffer, pH 6.8. The enzyme was then eluted as a broad peak with 0.25 M potassium phosphate buffer, pH 6.8. (e) Chromatography on L-leucine-Sepharose. The fractions from the hydroxyapatite step containing most of the activity were pooled and brought to 1 M
AND
KOHLHAW
ammonium sulfate by the addition of solid ammonium sulfate. Batches containing up to 200 mg of protein were then pumped onto a 2.5 x 30-cm column of L-leucine-Sepharose equilibrated with 1.0 M potassium phosphate buffer, pH 6.8. The column, with the enzyme bound, was then washed successively with 100 ml each of 1.0, 0.75, and 0.60 M potassium phosphate buffer, pH 6.8, and the enzyme was eluted with 0.4 M potassium phosphate buffer, pH 6.8. (fj Ammonium sulfate fractionation. Those fractions of the previous step containing approximately 80% of the eluted activity were concentrated by ultrafiltration through a PM-30 Diaflo membrane (Amicon) to approx I/5 of the original volume and precipitated by slowly adding solid ammonium sulfate to 65% saturation. The precipitated protein was redissolved in 0.1 M potassium phosphate buffer, pH 6.8, containing 20% glycerol and 0.02% sodium azide, and dialyzed against two changes of 1 liter each of the same buffer, over a 24-h period. The purified enzyme could be stored at 4°C for several weeks without loss of activity. The degree of purity was routinely determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis following the procedure of Fairbanks et al. (17). Preparation of amino acid-substituted Sepharose. Amino acid-Sepharoses were prepared essentially as described by Cuatrecasas and Aniinsen (18) by COUpling the desired amino acid directly to cyanogen bromide-activated Sepharose 4B (Pharmacial. The final products contained approximately 15 pmol of amino acid/ml of settled gel. Treatment of kinetic data. Some of the kinetic data in this paper are presented as double reciprocal plots. This is done because such plots are the most common method for determining inhibition patterns. However, such plots are notoriously poor when used to determine kinetic constants (191. Therefore, kinetic constants were calculated by weighted least-squares analysis utilizing a FORTRAN IV computer program (20, 211. Analytical ultracentrifugation. A Spinco model E analytical ultracentrifuge was used. The instrument was equipped with both Schlieren and Rayleigh interference optics and electronic speed control. Sedimentation equilibrium experiments were performed utilizing the long-column meniscus depletion technique of Chervenka (22). Samples were determined to be in equilibrium when a fringe displacement greater than 900 pm, measured at a fixed distance from the center of rotation, did not vary by more than 10 pm when photos were taken 3 h apart. The cells used in these experiments contained a 2l/2 a aluminum-filled Epon double sector capillary synthetic boundary centerpiece and were equipped with sapphire windows. Prior to a run, samples were dialyzed against 0.05 M sodium phosphate buffer, pH 7.00, containing 0.2 M sodium chloride.
FEEDBACK
INHIBITION
The dialysate was used as reference solution. Photographs were taken on Eastman Kodak II-G Spectroscopic plates. The plates were developed at room temperature in high contrast Eastman Kodak D-19 developer and fixed in Edwal’s Quick-fix. They were then read using a Nikon Microcomparator. RESULTS
Enzyme purification. Utilization of combined hydrophobic and affznity chromatography. Table I shows the results of a typical purification of feedback-resistant cY-isopropylmalate synthase. This procedure is an extensive modification of the one used before for the isolation of wildtype enzyme (2, 23). The purification of wild-type enzyme had made successful use of a leucine-induced shift in apparent molecular weight from about 150,000 to about 100,000 as measured by gel filtration, No such step could be used with the feedbackresistant enzyme which is not dissociated by leucine (8). In order to introduce specificity into the purification, use was made of a method of combined hydrophobic and affinity chromatography based on earlier observations (24) that both the feedbackresistant and wild-type a-isopropylmalate synthase bind to r,-1eucineSepharose at high concentrations (> 1.0 M) but not at low concentrations (< 0.2 M) of antichaotropic ions such as phosphate or sulfate, and that at intermediate concentrations of these ions, the interaction between both enzymes and cleucine-Sepharose is weak but rather specific. Thus, the a-isopropylmalate synthases as well as other proteins were strongly retained on L-leucine-Sepharose columns equilibrated with 1.0 M poTABLE PURIFICATION
OF FEEDBACK-RESISTANT
Step
Crude extract” Streptomycin sulfate precipitation Polyethylene glycol fractionation Hydroxyapatite (pool) L-leucine-Sepharose (pool) Ammonium sulfate precipitation
tassium phosphate buffer, pH 6.8. At this phosphate concentration, both enzymes also bound to nleucine-, n-isoleucine-, and n-valine-Sepharose. These interactions were probably at least in part due to hydrophobic effects (25-27). As the potassium phosphate concentration was lowered to 0.4 M, retention on n-leucine- and LvalineSepharose was essentially lost while L-leutine- and L-isoleucine-Sepharoses still weakly retained the two a-isopropylmalate synthases but not many extraneous proteins. (As will be shown below, both enzymes have sites for L-leucine and Lisoleucine) . The final specific activity of purified feedback-resistant cu-isopropylmalate synthase was very similar to that of highly purified wild-type enzyme, i.e., between 800 and 1000 (at the pH optimum of 8.5). Figure 1 shows a densitometer scan obtained after polyacrylamide gel electrophoresis in sodium dodecyl sulfate of feedback-resistant enzyme with specific activity of 810. The purity of this preparation was 85%. None of the impurities present amounted to more than 3% of the total protein. Upon gel filtration on Sephadex G-150, the purified material gave a symmetrical peak with coinciding activity and protein curves. Affinity for substrates and subktrate specificity. A previous report contains kinetic data pertaining to wild-type enzyme which were collected at pH 8.5 (2). At this pH value, both wild-type and feedbackresistant a-isopropylmalate synthase exhibit optimal activity. For the purpose of comparing the kinetic properties of the two I
(z-ISOPROPYLMALATE
Total activity
70,000 63,900 58,000 37,800 18,300 13,700
365
BY LEUCINE
SYNTHASE
Total protein (mg)
Specifi@ activity
6,550 6,310 2,040 193 27 17
10.7 10.1 28.5 197 660 810
FROM STRAIN
Recovery of activity (%I 100 91 83 54 26.5 19.5
CV-241 Fold purification 1.0 0.95 2.6 18.5 62.0 75.7
a From 52 g (wet weight) of cells suspended in 52 ml of 0.1 M potassium phosphate buffer, pH 6.8. See Methods for detailed description of each step. b Assays during purification were performed in 200 mM Tris.HCl buffer, pH 8.5, containing 80 mM KCl.
SOPER,
DOELLGAST
AND
KOHLHAW
resistant and the wild-type enzyme, respectively. Both enzymes exhibited rather 1 broad specificity for a-ketoacids but 0.5 showed no activity with a-ketodicarboxylic acids. cu-Ketobutyrate and pyruvate had turnover numbers higher than ob04served with the physiological substrate (Yketoisovalerate, but the apparent K, vals 0 0.3 ues for these two compounds were one and d two orders of magnitude higher, respectively, when compared with a-ketoisoval0.2 crate. In contrast to the broad specificity of the a-ketoisovalerate site, the acetyl-CoA 0.1 site had a very restricted specificity. Of the substrates tested, only acetyl-CoA was turned over by either enzyme. Acetyl-(3’0.JLL-h dephosphoj-CoA and the fluorescent anaI were not FIG. 1. Densitometer scan of polyacrylamide gel log acetyl-(1,N6-ethenoj-CoA turned over by either the wild-type or the obtained after electrophoresis of sodium dodecyl sulfeedback-resistant a-isopropylmalate synfate-denatured cy-isopropylmalate synthase purified from strain CV-241. The protein had been stained thase. Also, the next higher homolog of with Coomassie Blue. The gel was scanned at 550 acetyl-CoA, propionyl-CoA, which is a nm using a Gilford linear transport (Model 2410s) competitive inhibitor with respect to aceconnected to a Gilford spectrophotometer (Model tyl-CoA (26), was not turned over under 240). The arrow indicates the top of the gel. standard assay conditions. Specificity of inhibition. The inhibition enzymes, the pH was changed to 7.0, howof wild-type a-isopropylmalate synthase ever. This was done because of a strong by leucine is very specific (14). Of all natudesensitization against leucine inhibition ral amino acids tested, only leucine and to at higher pH values (2, and unpublished a much lesser extent isoleucine, but not observations) rendering a study of leucine In view of the inhibition of the feedback-resistant en- valine, were inhibitory. strong desensitization of the feedback-rezyme at pH values above 7.5 practically sistant enzyme to leucine inhibition, it impossible. Also, a number of binding and structural studies with wild-type enzyme was of interest to find out whether a similar desensitization had occurred with rewere performed at or near neutrality ($8). spect to isoleucine inhibition. Table II With respect to substrate affinity and shows that this was not the case. The feedspecificity, the only significant difference back-resistant enzyme was actually more between the two enzymes was found with sensitive to isoleucine and its analog, the apparent K, value for o-ketoisovalerthiaisoleucine, than was wild-type enate. Under standard assay conditions and zyme. This surprising result was not with a final enzyme concentration” of 2 pgl caused by a shift in the pH dependence of ml, Km, app was 22 -+ 10 (SE) PM for the which was very simifeedback-resistant and 69 ? 18 (SE) PM for isoleucine inhibition, lar with both enzymes. In addition to the the wild-type enzyme. The apparent K, compounds listed in Table II, the following values for acetyl-CoA were 210 f 30 (SE) amino acids or derivatives when tested at PM and 250 + 90 (SE) PM for the feedbacka concentration of 5 mM had no effect on 6 Preliminary results indicate that various kithe activity of either enzyme: N-methylnetic constants of a-isopropylmalate synthase are n,cleucine, N-methyl-n,L-isoleucine, Lenzyme concentration-dependent. This must be leucine amide, L-isoleucine amide, nleutaken into account when comparing the numerical L-threonine, L-alanine, values of separate experiments. Care was taken to tine, n-isoleucine, cglutamine, L-histidine, LLasparate , keep the final protein concentration constant in any phenylalanine, Irtryptophan, and L-lyone set of experiments. 06-
TOP
FEEDBACK TABLE
INHIBITION
II
SPECIFICITY OF INHIBITION OF WILD-TYPE AND FEEDBACK-RESISTANT QL-ISOPROPYLMALATE SYNTHASE BY AMINO ACIDS AND ANALOGS Addition”
Inhibition Wild-type enzyme
L-leucine, 0.05 mM L-leucine, 5 mM 5’,5’,5-trifluoroleucine, rnM Azaleucine, 5 mM L-isoleucine, 5 mM Thiaisoleucine, 5 mM L-valine, 5 mM
5
(o/o) Feedbackresistant enzyme
75 >95 95
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Mechanism Purification THOMAS
and Properties
of Feedback
Inhibition
of a Feedback-Resistant
S. SOPER2, GEORGE Department
173, 362-374 (1976)
J. DOELLGAST3,
of Biochemistry,
Purdue Received
University, August
by Leucine c&opropylmalate
AND
GUNTER
West Lafayette,
Indiana
Synthase’
B. KOHLHAW4 47907
11, 1975
A mutationally altered, n-leucine-resistant form of a-isopropylmalate synthase, the first committed enzyme in leucine biosynthesis, has been purified to near homogeneity. Comparison of the feedback-resistant enzyme with its wild-type parent shows the following: Both enzymes are very similar with respect to substrate specificity and maximal activity, but the feedback-resistant enzyme has a greater affinity for one of the substrates, a-ketoisovalerate. The feedback-resistant enzyme is about three orders of magnitude less sensitive to L-leucine than wild-type enzyme. By contrast, it is slightly more sensitive to r,-isoleucine, the only other naturally occurring amino acid known to inhibit a-isopropylmalate synthase. Results of chemical densensitization experiments suggest that the leucine, isoleucine, and active sites are distinct. The kinetic pattern of leucine inhibition at pH 7.0 shows that leucine is a noncompetitive inhibitor with respect to both substrates with wild-type enzyme, whereas the weak inhibition by leucine of the feedback-resistant enzyme is of a competitive type. Intersubunit cross-linking of the feedback-resistant enzyme followed by gel electrophoresis in sodium dodecyl sulfate reveals the presence of monomers, dimers, and tetramers with molecular weights of approximately 52,000, 110,000, and 200,000, respectively. Very similar results had been obtained with wild-type enzyme. Sedimentation equilibrium analyses indicate that both enzymes exist as associating-dissociating systems that can be adequately described by either a monomer-tetramer or a monomer-dimer-tetramer equilibrium. With the feedback-resistant enzyme, the equilibrium constant for the monomer-tetramer equilibrium. K, = [A,]/[A14, is 1 x 10Ly M-~, compared with 9 x 10’” Mm3 for wild-type enzyme. This suggests a stronger tendency of the subunits of the feedback-resistant enzyme to aggregate, a conclusion supported by gel filtration experiments. These results, together with previous observations that wild-type enzyme is dissociated by leucine whereas the feedback-resistant enzyme is not, suggest that efficient inhibition of cY-isopropylmalate synthase by leucine may be coupled to a relatively loose arrangement of subunits within the oligomeric structure of the enzyme.
The leucine biosynthetic pathway in Salmonella typhimurium consists of three unique enzymes: cY-isopropylmalate syn-
thase (EC 4.1.3.12), isopropylmalate isomerase (EC 4.2.1.33), and /3-isopropylmalate dehydrogenase (EC 1.1.1.85). The product of these reactions, cr-ketoisocaproate, is converted to leucine by transaminase B (EC 2.6.1.6). The pathway is controlled by both end-product repression and end-product (feedback) inhibition (l-3). Feedback inhibition by leucine is directed toward the first enzyme in the pathway, which catalyzes the condensation of acetylCoA and cr-ketoisovalerate to give a-isopropylmalate and CoA. We have previ-
’ This work was supported by Research Grant GM 15102 from the National Institute of General Medical Sciences. Journal Paper No. 5992 of the Agricultural Experiment Station, Purdue University. * Present address: Rockefeller University, New York, N. Y. 10021. 3 Present address: Tufts University, School of Medicine, 136 Harrison Ave., Boston, Mass. 02111. *To whom all correspondence should be addressed. 362 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
FEEDBACK
INHIBITION
ously reported that wild-type a-isopropylmalate synthase has a subunit molecular weight of 50,000 +- 3000 (4, 51, and that, in the absence of ligands, the enzyme exists in a rapid association-dissociation equilibrium between monomers, tetramers, and possibly dimers (5). The presence of one substrate together with an analog of the other favors formation of tetramers (5) while the presence of the feedback inhibitor causes dissociation, usually leading to a predominance of dimers (2, 4, 6, 7). The stabilization of dimers by leucine is probably related to the fact that there is only one saturable leucine binding site per dimer (8). The availability of a strain producing constitutively high levels of a feedbackresistant a-isopropylmalate synthase (strain CV-241; 9) made it possible to embark on an extensive comparison of this enzyme with wild-type cY-isopropylmalate synthase and, thus, opened a new avenue for the study of leucine inhibition. It has already been established that the feedback-resistant enzyme is approximately three orders of magnitude less sensitive to leucine than its wild-type counterpart. Thus, in 80 mM potassium phosphate buffer, pH 6.8, and in the presence of 0.8 mM acetyl-CoA and 4 mM a-ketoisovalerate, the apparent K i value was found to be 20 mM for the feedback-resistant and 22 PM for wild-type enzyme (8). It has also been shown that the feedback-resistant enzyme is not dissociated by leucine, even when inhibitory concentrations of the amino acid are employed (2, 8, 10). This suggested that the mutation to feedback resistance has led to changes in the quaternary structure of the enzyme. We report in this paper that wild-type and feedback-resistant a-isopropylmalate synthases of comparable purity exhibit several subtle but important differences in addition to the large difference in leucine sensitivity. Of particular significance are results that indicate that the feedback-resistant enzyme apparently still exists in an equilibrium involving monomers, dimers, and tetramers, but that it has a greater tendency to aggregate within the framework of this equilibrium when compared with wild-type enzyme. We discuss
363
BY LEUCINE
these results in terms of an inverse relationship between the strength of subunit interactions and the extent of inhibition by leucine. EXPERIMENTAL
PROCEDURE
Materials Chemical. Special chemicals and their suppliers were: i,-leucine, L-isoleucine, n-leucine, n-isoleutine, N-methyl-n,n-leucine, n-leucine amide, and pyridoxal-5’-phosphate, Sigma; L-valine, N-methylD,L-isoleucine, a-ketoisovalerate, and 5,5’-dithiobis(2-nitrobenzoate), Calbiochem; L-isoleucine amide, Cycle; CoA (lithium salt, “chromatopure,” 90% reduced CoA), 3’-dephospho-CoA and (l,N”-ethenojCoA, PL-Biochemicals; Tris5 base, enzyme grade, Schwart=Mann; polyethyland urea, “ultrapure,” ene glycol 6000 (molecular weights, 600&7500), Matheson, Coleman and Bell. All other commercially obtained chemicals were of the best available grade. 5’,5’,5’-trifluoroleucine, azaleucine, and thiaisoleucine were gifts from H. E. Umbarger, Purdue University. A generous gift of trifluoroleucine was also obtained from H. S. Anker and D. F. Steiner, University of Chicago. Acetyl-CoA and propionyl-CoA were synthesized from CoA and the appropriate acid anhydride, following the general procedure of Simon and Shemin (11). Dimethylsuberimidate was prepared according to Davies and Stark
(12). Biological. The two strains utilized are both derivatives of Salmonella typhimurium LT-2. Strain CV-19 (genotype ara-9 gal-205 flrB), isolated as a 5’,5’,5’-trifluoroleucine-resistant mutant, served as the source for wild-type enzyme. This strain carries a mutation unlinked to the leucine operon CjlrB; 13) which results in constitutively high levels of the leucine as well as the isoleucine-valine biosynthetic enzymes (14). Strain CV-241 (genotype 1euA 2010 gal-205 flrB) served as the source of feedback-resistant enzyme. It was the result of a transduction cross (91, and carries the flrB marker and, in addition, a mutation in the structural gene for a-isopropylmalate synthase (leaA 2010), which renders this enzyme much less sensitive to inhibition by leucine (8, 14). Cells were grown and harvested as described previously (2). Methods Assay for a-isopropylmalate synthase. An endpoint assay based on the determination of free CoA with 5,5’-dithiobis-(2-nitrobenzoate) was used throughout this study. The standard assay mixture contained potassium phosphate buffer, pH 7.0, 200 s Abbreviation aminomethane.
used: Tris,
tris-(hydroxymethyl)-
364
SOPER,
DOELLGAST
mM; acetyl-CoA, 0.8 mM; a-ketoisovalerate, 4.0 mbr; and enzyme to give a final absorbance of no more than 0.3 units at 412 nm and 37°C. The assay volume was 0.125 ml. Under these conditions, the assay was linear with time for at least 10 min. The reaction was stopped by addition of 3 vol of ethanol. To this were added 2 vol of a 1 mM solution of 5,5’-dithiobis(2-nitrobenzoate) in 50 rnM Tris-HCl buffer, pH 7.5. The molar extinction coefficient of the 5-carboxy-4nitrothiophenolate ion formed is 13,600 at 412 nm (15). Specific activity of the enzyme is defined as *moles of CoA formed per h per mg of protein. Protein determination. Protein was determined by the biuret method (16) with bovine serum albumin as standard. Interference by glycerol was corrected for with appropriate standard curves. Purification of feedback-resistant a-isopropylmalate synthase. (a) Preparation of crude extract. A 1:l (w/v) suspension of cells in 0.1 M potassium phosphate buffer, pH 6.8, was passed through an Aminco French’Pressure Cell in 40-ml portions, at a pressure of 540 atm. The suspension was then diluted with 2 parts of the same buffer and centrifuged at 48,OOOgfor 1 h. The pellet was discarded. (b) Streptomycin sulfate precipitation. An amount of streptomycin sulfate equal to 25% of the total protein (by weight), applied as a 10% (w/v) solution in 0.1 M potassium phosphate, pH 6.8, was slowly added to the crude extract. The extract was then centrifuged at 28,000g for 30 min, and the pellet discarded. (c) Fractionation zuith polyethylene glycol. The protein concentration was adjusted to 16 to 20 mg/ml by addition of 0.1 M potassium phosphate buffer, pH 6.8. Granulated polyethylene glycol6000 was slowly added to give a concentration of 6% (w/v). The precipitate was collected by centrifugation for 10 min at 10,OOOg and dissolved in the aforementioned buffer to give a solution containing no less than 10 mg of protein/ml. (d) Hydroxyapatite chromatography. One milliliter of hydroxyapatite suspension (made up in 0.1 M phosphate buffer and measured when settled) per 50 mg of protein was added to the solution obtained in the previous step. The suspension was stirred for 1 h at a slow speed (sufficient to keep the hydroxyapatite in suspension) and centrifuged for 10 min at 150g. The pellet was washed three times with 0.16 M potassium phosphate buffer, pH 6.8, resuspended in the same buffer, and poured into a glass column which already contained 1 ml of settled hydroxyapatitel2 mg of protein. The added hydroxyapatite was allowed to settle, and was washed with two column volumes of 0.16 M potassium phosphate buffer, pH 6.8. The enzyme was then eluted as a broad peak with 0.25 M potassium phosphate buffer, pH 6.8. (e) Chromatography on L-leucine-Sepharose. The fractions from the hydroxyapatite step containing most of the activity were pooled and brought to 1 M
AND
KOHLHAW
ammonium sulfate by the addition of solid ammonium sulfate. Batches containing up to 200 mg of protein were then pumped onto a 2.5 x 30-cm column of L-leucine-Sepharose equilibrated with 1.0 M potassium phosphate buffer, pH 6.8. The column, with the enzyme bound, was then washed successively with 100 ml each of 1.0, 0.75, and 0.60 M potassium phosphate buffer, pH 6.8, and the enzyme was eluted with 0.4 M potassium phosphate buffer, pH 6.8. (fj Ammonium sulfate fractionation. Those fractions of the previous step containing approximately 80% of the eluted activity were concentrated by ultrafiltration through a PM-30 Diaflo membrane (Amicon) to approx I/5 of the original volume and precipitated by slowly adding solid ammonium sulfate to 65% saturation. The precipitated protein was redissolved in 0.1 M potassium phosphate buffer, pH 6.8, containing 20% glycerol and 0.02% sodium azide, and dialyzed against two changes of 1 liter each of the same buffer, over a 24-h period. The purified enzyme could be stored at 4°C for several weeks without loss of activity. The degree of purity was routinely determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis following the procedure of Fairbanks et al. (17). Preparation of amino acid-substituted Sepharose. Amino acid-Sepharoses were prepared essentially as described by Cuatrecasas and Aniinsen (18) by COUpling the desired amino acid directly to cyanogen bromide-activated Sepharose 4B (Pharmacial. The final products contained approximately 15 pmol of amino acid/ml of settled gel. Treatment of kinetic data. Some of the kinetic data in this paper are presented as double reciprocal plots. This is done because such plots are the most common method for determining inhibition patterns. However, such plots are notoriously poor when used to determine kinetic constants (191. Therefore, kinetic constants were calculated by weighted least-squares analysis utilizing a FORTRAN IV computer program (20, 211. Analytical ultracentrifugation. A Spinco model E analytical ultracentrifuge was used. The instrument was equipped with both Schlieren and Rayleigh interference optics and electronic speed control. Sedimentation equilibrium experiments were performed utilizing the long-column meniscus depletion technique of Chervenka (22). Samples were determined to be in equilibrium when a fringe displacement greater than 900 pm, measured at a fixed distance from the center of rotation, did not vary by more than 10 pm when photos were taken 3 h apart. The cells used in these experiments contained a 2l/2 a aluminum-filled Epon double sector capillary synthetic boundary centerpiece and were equipped with sapphire windows. Prior to a run, samples were dialyzed against 0.05 M sodium phosphate buffer, pH 7.00, containing 0.2 M sodium chloride.
FEEDBACK
INHIBITION
The dialysate was used as reference solution. Photographs were taken on Eastman Kodak II-G Spectroscopic plates. The plates were developed at room temperature in high contrast Eastman Kodak D-19 developer and fixed in Edwal’s Quick-fix. They were then read using a Nikon Microcomparator. RESULTS
Enzyme purification. Utilization of combined hydrophobic and affznity chromatography. Table I shows the results of a typical purification of feedback-resistant cY-isopropylmalate synthase. This procedure is an extensive modification of the one used before for the isolation of wildtype enzyme (2, 23). The purification of wild-type enzyme had made successful use of a leucine-induced shift in apparent molecular weight from about 150,000 to about 100,000 as measured by gel filtration, No such step could be used with the feedbackresistant enzyme which is not dissociated by leucine (8). In order to introduce specificity into the purification, use was made of a method of combined hydrophobic and affinity chromatography based on earlier observations (24) that both the feedbackresistant and wild-type a-isopropylmalate synthase bind to r,-1eucineSepharose at high concentrations (> 1.0 M) but not at low concentrations (< 0.2 M) of antichaotropic ions such as phosphate or sulfate, and that at intermediate concentrations of these ions, the interaction between both enzymes and cleucine-Sepharose is weak but rather specific. Thus, the a-isopropylmalate synthases as well as other proteins were strongly retained on L-leucine-Sepharose columns equilibrated with 1.0 M poTABLE PURIFICATION
OF FEEDBACK-RESISTANT
Step
Crude extract” Streptomycin sulfate precipitation Polyethylene glycol fractionation Hydroxyapatite (pool) L-leucine-Sepharose (pool) Ammonium sulfate precipitation
tassium phosphate buffer, pH 6.8. At this phosphate concentration, both enzymes also bound to nleucine-, n-isoleucine-, and n-valine-Sepharose. These interactions were probably at least in part due to hydrophobic effects (25-27). As the potassium phosphate concentration was lowered to 0.4 M, retention on n-leucine- and LvalineSepharose was essentially lost while L-leutine- and L-isoleucine-Sepharoses still weakly retained the two a-isopropylmalate synthases but not many extraneous proteins. (As will be shown below, both enzymes have sites for L-leucine and Lisoleucine) . The final specific activity of purified feedback-resistant cu-isopropylmalate synthase was very similar to that of highly purified wild-type enzyme, i.e., between 800 and 1000 (at the pH optimum of 8.5). Figure 1 shows a densitometer scan obtained after polyacrylamide gel electrophoresis in sodium dodecyl sulfate of feedback-resistant enzyme with specific activity of 810. The purity of this preparation was 85%. None of the impurities present amounted to more than 3% of the total protein. Upon gel filtration on Sephadex G-150, the purified material gave a symmetrical peak with coinciding activity and protein curves. Affinity for substrates and subktrate specificity. A previous report contains kinetic data pertaining to wild-type enzyme which were collected at pH 8.5 (2). At this pH value, both wild-type and feedbackresistant a-isopropylmalate synthase exhibit optimal activity. For the purpose of comparing the kinetic properties of the two I
(z-ISOPROPYLMALATE
Total activity
70,000 63,900 58,000 37,800 18,300 13,700
365
BY LEUCINE
SYNTHASE
Total protein (mg)
Specifi@ activity
6,550 6,310 2,040 193 27 17
10.7 10.1 28.5 197 660 810
FROM STRAIN
Recovery of activity (%I 100 91 83 54 26.5 19.5
CV-241 Fold purification 1.0 0.95 2.6 18.5 62.0 75.7
a From 52 g (wet weight) of cells suspended in 52 ml of 0.1 M potassium phosphate buffer, pH 6.8. See Methods for detailed description of each step. b Assays during purification were performed in 200 mM Tris.HCl buffer, pH 8.5, containing 80 mM KCl.
SOPER,
DOELLGAST
AND
KOHLHAW
resistant and the wild-type enzyme, respectively. Both enzymes exhibited rather 1 broad specificity for a-ketoacids but 0.5 showed no activity with a-ketodicarboxylic acids. cu-Ketobutyrate and pyruvate had turnover numbers higher than ob04served with the physiological substrate (Yketoisovalerate, but the apparent K, vals 0 0.3 ues for these two compounds were one and d two orders of magnitude higher, respectively, when compared with a-ketoisoval0.2 crate. In contrast to the broad specificity of the a-ketoisovalerate site, the acetyl-CoA 0.1 site had a very restricted specificity. Of the substrates tested, only acetyl-CoA was turned over by either enzyme. Acetyl-(3’0.JLL-h dephosphoj-CoA and the fluorescent anaI were not FIG. 1. Densitometer scan of polyacrylamide gel log acetyl-(1,N6-ethenoj-CoA turned over by either the wild-type or the obtained after electrophoresis of sodium dodecyl sulfeedback-resistant a-isopropylmalate synfate-denatured cy-isopropylmalate synthase purified from strain CV-241. The protein had been stained thase. Also, the next higher homolog of with Coomassie Blue. The gel was scanned at 550 acetyl-CoA, propionyl-CoA, which is a nm using a Gilford linear transport (Model 2410s) competitive inhibitor with respect to aceconnected to a Gilford spectrophotometer (Model tyl-CoA (26), was not turned over under 240). The arrow indicates the top of the gel. standard assay conditions. Specificity of inhibition. The inhibition enzymes, the pH was changed to 7.0, howof wild-type a-isopropylmalate synthase ever. This was done because of a strong by leucine is very specific (14). Of all natudesensitization against leucine inhibition ral amino acids tested, only leucine and to at higher pH values (2, and unpublished a much lesser extent isoleucine, but not observations) rendering a study of leucine In view of the inhibition of the feedback-resistant en- valine, were inhibitory. strong desensitization of the feedback-rezyme at pH values above 7.5 practically sistant enzyme to leucine inhibition, it impossible. Also, a number of binding and structural studies with wild-type enzyme was of interest to find out whether a similar desensitization had occurred with rewere performed at or near neutrality ($8). spect to isoleucine inhibition. Table II With respect to substrate affinity and shows that this was not the case. The feedspecificity, the only significant difference back-resistant enzyme was actually more between the two enzymes was found with sensitive to isoleucine and its analog, the apparent K, value for o-ketoisovalerthiaisoleucine, than was wild-type enate. Under standard assay conditions and zyme. This surprising result was not with a final enzyme concentration” of 2 pgl caused by a shift in the pH dependence of ml, Km, app was 22 -+ 10 (SE) PM for the which was very simifeedback-resistant and 69 ? 18 (SE) PM for isoleucine inhibition, lar with both enzymes. In addition to the the wild-type enzyme. The apparent K, compounds listed in Table II, the following values for acetyl-CoA were 210 f 30 (SE) amino acids or derivatives when tested at PM and 250 + 90 (SE) PM for the feedbacka concentration of 5 mM had no effect on 6 Preliminary results indicate that various kithe activity of either enzyme: N-methylnetic constants of a-isopropylmalate synthase are n,cleucine, N-methyl-n,L-isoleucine, Lenzyme concentration-dependent. This must be leucine amide, L-isoleucine amide, nleutaken into account when comparing the numerical L-threonine, L-alanine, values of separate experiments. Care was taken to tine, n-isoleucine, cglutamine, L-histidine, LLasparate , keep the final protein concentration constant in any phenylalanine, Irtryptophan, and L-lyone set of experiments. 06-
TOP
FEEDBACK TABLE
INHIBITION
II
SPECIFICITY OF INHIBITION OF WILD-TYPE AND FEEDBACK-RESISTANT QL-ISOPROPYLMALATE SYNTHASE BY AMINO ACIDS AND ANALOGS Addition”
Inhibition Wild-type enzyme
L-leucine, 0.05 mM L-leucine, 5 mM 5’,5’,5-trifluoroleucine, rnM Azaleucine, 5 mM L-isoleucine, 5 mM Thiaisoleucine, 5 mM L-valine, 5 mM
5
(o/o) Feedbackresistant enzyme
75 >95 95
