The substrate specificity of glutamic acid dehydrogenase

ARCHIVES

OF

BIOCHEMISTRY

The Substrate

AND

BIOPHYSICS

Specificity

JACOB STRUCK,

86, 260-266 (1960)

of Glutamic JR.’

From the Division of Biochemistry, Institute of Technology,

Acid

AND IRWIN Department Cambridge,

Dehydrogenase

W. SIZER

of Biology, Massachusetts Massachusetts

Received August 10, 1959 An enzyme present in chicken liver, beef liver, and pig kidney has been found to catalyze a diphosphopyridine nucleotide (DPN)-dependent oxidation of n-leucine. Crystalline n-glutamic acid dehydrogenase is found to bring about the same reaction. The data obtained so far support the contention that the oxidation of leucine, as well as other n-a-amino acids, may be attributed to the nonspecific action of n-glutamic acid dehydrogenase. Its optimum pH varies with the substrate and is 8.4 for glutamate but 9.8 for leucine.

INTRODUCTION The wide distribution of L-glutamic acid microorganisms, among dehydrogenase plants, and animals suggests the importance of the reversible deamination of glutamic acid in metabolism. Aside from the alanine dehydrogenase, which has been reported to occur in a mutant of Bacillus subtilis (l), glutamic acid dehydrogenase appears to be the only enzyme which catalyzes a pyridine nucleotide-linked oxidation of an amino acid. The reversibility of the deamination provides a major enzymic mechanism for the interconversion of cY-keto and amino acids coupled with the formation or fixation of ammonia. In view of the apparent metabolic significance of this enzyme, it is noteworthy that previous examination of the substrate specificity of glutamic acid dehydrogenase failed to detect an oxidation of other amino acids (2-4). We wish to report evidence that under appropriate conditions glutamic acid dehydrogenase can catalyze the oxidation of certain other L-a-amino acids. EXPERIMENTqL n-Glutamic acid was supplied as the monosodium salt by Mann Research Laboratories. Aque-

ous solutions of bovine plasma albumin (Pentex, Inc.) were dialyzed against distilled water and stored frozen. Reduced diphosphopyridine nucleotide (DPNH) (Pabst Laboratories) was dissolved in distilled water and adjusted to pH 859.0 with dilute sodium hydroxide. The concentration of

DPNH in these solutions was determined from absorbance measurements at 340 and 260 mp. o-Ketoisocaproic acid was synthesized by the method of Adickes and Andresen (5). Crystalline glutamic acid dehydrogenase was supplied by Nutritional Biochemicals Corporation as a suspension in 2.1 M ammonium sulfate. A single lot of 500 mg. was used in all of the experiments described below. Solutions of the enzyme were routinely prepared in 0.05 M potassium phosphate buffer, pH 7.4, and extensively dialyzed against the same buffer in order to remove ammonium sulfate. The concentration of crystalline enzyme was estimated from measurements of the absorbance of solutions at 270 rnp using the extinction coefficient reported by Olson and Anfinsen (3). Results of such protein determintions were identical with the biuret assay described by Mokrasch and McGilvery (6). The protein concentration of crude enzyme preparations was determined with the biuret reagent after a calibration curve was prepared using plasma albumin.

PREPARATION OF MITOCHONDRIAL ACETONE POWDERS

1 Present address : Miles Chemical Company, Division of Miles Laboratories, Inc., Clifton, New Jersey.

Fresh chicken livers were purchased from a local poultry dealer, and preparation of the acetone powders was carried out immediately. All opera260

261

GLUTAMIC ACID DEHYDROGENASE tions were performed at O-4%. unless otherwise stated, and all apparatus and solutions were chilled in the cold room. Livers were minced by a single passage through a meat grinder. One-hundred-gram portions of ground tissue were suspended in 300 ml. of a solution containing 85 g. sucrose and 5 g. of dibasic potassium phosphate per liter. The suspension was homogenized for 30 sec. in a Waring blendor operated from a powerstat set at 60 v. The homogenate was then centrifuged in an International refrigerated centrifuge (model PR-1) for 5 min. at 2400 r.p.m. The supernatant fluid, which contained the mitochondria and microsomes, was decanted through a single layer of cheesecloth, and the residue was discarded. To the supernatant fluid was added one. tenth its volume of 1.5 M potassium chloride. The mixture was stirred for 10 min. and then centrifuged for 30 min. at 5300 X g in a Stock centrifuge (type 6 X 1350). The sedimented material obtained from 6 to 7 1. of suspension in potassium chloride was resuspended in 1 1. of distilled water by a brief treatment in the Waring blendor. This suspension was poured into 10 vol. acetone at -20°C. and stirred vigorously for 15-29 min. The precipitate was separated on a Biichner funnel and then treated in a Waring blendor with approximately 10 vol. of cold acetone. Again the mixture was filtered on a Btichner funnel, and the precipitate was washed on the filter with cold ethyl ether. The fine powder obtained was air-dried at room temperature for l-2 hr. and then further dried in DUCUOover phosphorus pentoxide. This procedure yielded 20-30 g. of a tan powder/kg. of fresh tissue.

ENZYME ASSAY Leucine dehydrogenase was assayed by measuring the change in absorbance at 340 rnp which occurs as a result of the formation of DPNH. Measurement of absorbance was made with a Beckman DU spectrophotometer. A l-cm. cuvette was prepared with 300 pmoles tris(hydroxymethyl)aminomethane (Tris), pH 9.0, 50 rmoles L-leucine, and 3 pmoles diphosphopyridine nucleotide (DPN) in 2.9 ml. of a solution containing 0.05% albumin. At zero time, 0.10 ml. of properly diluted enzyme solution was added from an adder-mixer of the type described by Boyer and Segal (7). The absorbance at 340 rnp was recorded at 30-sec. intervals for at least 4 min. Absorbance readings were plotted against time, and the initial rate of the reaction was determined from the slope of the graph. Curves were found to be linear for at least 4 min., if the rate of change of absorbance did not exceed O.OB/min. Rate was proportional to enzyme concentration under these conditions. All experiments were carried out at 22-25°C. Rates were cor-

rected for blank determinations absence of substrate. An identical procedure was tamic acid dehydrogenase. In the pH of the Tris buffer was L-glutamic acid was employed

performed

in the

used to assay gluthis case, however, 8.0, and 50 rmoles as substrate.

RESULTS

PARTIAL

PURIFICATION OF L-LEUCINE DEHYDROGENASE

Although L-leucine is oxidized by both microsomal and mitochondrial fractions of chicken liver, the enzymic mechanism operating in each fraction appeared to differ. A preliminary report of the activity of the microsome fraction has appeared (8). Extracts of mitochondria were found to catalyze a DPN-dependent oxidation of L-leucine which was linearly related to enzyme concentration. The reaction was readily reversible in the presence of DPNH, ol-ketoisocaproic acid, and ammonia. The rate of the reverse reaction also increases linearly with enzyme concentration. Leucine oxidation was catalyzed by an enzyme present in extracts of acetone powders prepared from mitochondria of chicken liver, beef liver, and pig kidney. During attempts to purify the chicken liver enzyme from mitochondrial acetone powders, it was noted that glutamic acid dehydrogenase was present in all fractions which contained leucine dehydrogenase. Furthermore, the ratio of the rates of oxidation of glutamic acid and leucine remained constant in all fractions examined. This observation suggested the possibility that the oxidation of leucine could be attributed to the nonspecific action of glutamic acid dehydrogenase. Therefore, a fractionation procedure was carried out, and the ratio of glutamic acid to leucine dehydrogenase activities was measured on each fraction in order to determine whether or not any separation of the two activities had occurred. The fractionation procedure employed was as follows: Thirty grams of mitochondrial acetone powder was extracted by stirring with 300 ml. of 0.05 M potassium phosphate buffer, pH 7.4, for 60 min. at 4°C. The suspension was centrifuged for 15 min. at 10,000 r.p.m. in a Lourdes centrifuge (model AA-l).

262

STRUCK AND SIZER TABLE

I

THE RATIO OF L-GLUTAMIC ACID TO L-LEUCINE DEHYDROGENASE ACTIVITY DURING FRACTIONATION Enzymes were assayed as described under Experimental. Specific activities are expressed as the change in absorbance/min./mg. protein/3 ml. reaction mixture. Recoveries are based on assays performed with L-leucine as substrate.

-

I

Specific activity Fraction

Acetone powder extract Ethanol, O-20% pH precipitate Ammonium sulfate, 035% Ammonium sulfate, 3545% Centrifuged pellel

P s 2 %

I&2cin,

(2)

L-Glutamic acid

100

0.011

0.72

65

25 75 45

0.014 0.042 0.070

0.87 2.38 4.16

63 55 60

6

0.027

1.69

63

t 38 0.142 - - -

8.58

i

61

The supernatant solution was decanted and is referred to as the acetone powder extract. The extract was cooled to -5°C. during the addition of ethanol to a final concentration of 20% by volume. After 30 min. the precipitate was removed by centrifugation at -5”‘C. in the International centrifuge, then dissolved in 25 ml. of 0.05 M potassium phosphate buffer, pH 7.4, and finally dialyzed overnight against a large volume of the same buffer. This dialyzed solution is referred to as the O-20 % ethanol fraction. The supernatant solution from the centrifugation was stirred at -5”C., and 1 M acetate buffer, pH 5.5, was slowly added until the pH was 6.4-6.6. (Measurement of the pH with a Beckman pH meter, model G, was made after diluting the ethanol solution with 10 vol. water.) The mixture was stirred for an additional 30 min. The precipitate was removed by centrifugation in the refrigerated centrifuge and dissolved in 25 ml. of 0.05 M potassium phosphate buffer, pH 7.4. After dialysis against the same buffer for 12 hr., the fraction, referred to as the pH precipitate, was further fractionated with a saturated solution of ammonium sulfate. The saturated solution of

ammonium sulfate was prepared in distilled water, and the pH was adjusted to 7.4-7.6 with concentrated ammonium hydroxide. Since this solution was stored at 4”C., the degree of saturation refers to this temperature. Enough of this solution was added to the dialyzed pH-precipitated fraction to make the final concentration 35 % saturated with respect to ammonium sulfate. The precipitate was removed by centrifugation and dissolved in about 10 ml. of 0.05 M potassium phosphate buffer at pH 7.4. To the supernatant solution, ammonium sulfate was added to 45% saturation. The precipitate was collected by centrifugation and dissolved in 5 ml. of the phosphate buffer. Both ammonium sulfate fractions were dialyzed against this buffer in order to remove completely the ammonium sulfate. An additional purification of the dialyzed O-35% ammonium sulfate fraction was effected by subjecting this solution to centrifugation in a Spinco (model L) preparative ultracentrifuge. Centrifugation was carried out at 40,000 r.p.m. for 4 hr. in the No. 40 rotor. The gelatinous pellet was redissolved in 0.05 M potassium phosphate buffer, pH 7.4. A summary of the results of this fractionation procedure is presented in Table I. The final preparation contained 38 % of the original activity of the extract and represented a 12-fold purification. Examination of the data reveals that the ratio of activities determined with L-glutamic acid and Lleucine remained constant during the purification. THE OXIDATION OF L-LEUCINE BY CRYSTALLINE GLUTAMIC ACID DEHYDROGENASE

The constant ratio of activities during purification supports the hypothesis that the oxidation of n-leucine is the result of the nonspecific action of L-glutamic acid dehydrogenase. To test this possibility, crystalline beef liver glutamic acid dehydrogenase was examined for its ability to catalyze the oxidation of L-leucine. Figure 1 indicates that this enzyme did catalyze the oxidation of Lleucine and that the reaction was reversed by the addition of ammonia. Under the con-

GLUTAMIC

ACID

ditions employed, the ratio of the rate of oxidation of L-glutamic acid to that of Lleucine was approximately 60, which is similar to that observed with crude and purified fractions from chicken liver. Homogeneity of the crystalline enzyme was examined by boundary electrophoresis and by ultracentrifugation. Electrophoresis at an ionic strength of 0.1 in sodium phosphate buffer, pH 7.7, revealed only a single component. The ultracentrifuge pattern indicated the presence of a minor component (approximately 10% of the total) which sedimented less rapidly than the bulk of the protein. Since the difference between the rates of oxidation of the two substrates was such that a relatively small contamination of the crystalline glutamic acid dehydrogenase with another enzyme could account for L-leucine oxidation, attempts were then made to effect a separation of activities. The presence of two enzymes would be indicated by a change in the rates of oxidation of glutamic acid and leucine resulting from the separation procedures. If two enzymes were present, it should be possible to demonstrate a difference in their rate of sedimentation in an ultracentrifuge. To test this possibility, 2.0-ml. aliquots of a solution containing 2.02 mg. of the crystalline dehydrogenase/ml. in 0.05 M potassium phosphate buffer, pH 7.4, were centrifuged in the No. 40 rotor of the Spinco (model L) preparative ultracentrifuge equipped with microtube adapters. After a given period of centrifugation at 40,000 r.p.m., the supernatant solution was decanted and the gelatinous pellet was redissolved in 2.0 ml. of the buffer. Evidence for the separation of two different enzymes was sought by measurement of the activities of the materials remaining in the supernatant solution and those sedimented. Table II indicates that there was no separation by ultracentrifugation. Differential heat inactivation was also used in an attempt to demonstrate the presence of separate leucine and glutamic acid dehydrogenases. A solution of crystalline glutamic acid dehydrogenase (protein concentration of 5.87 mg./ml.) was prepared in 0.05 M potassium phosphate, pH

263

DEHYDROGENASE

0

40

120

80 TIME, MIN.

FIG. 1. The reduction of DPN in the presence of L-leucine by glutamic acid dehydrogenase and its reversal by ammonium chloride. The reaction mixture contained 300 pmoles Tris, pH 9.0, 0.3 pmole DPN, 151 pmoles L-leucine, and 0.58 mg. crystalline glutamic acid dehydrogenase in a total volume of 3.0 ml. After 80 min., 0.10 ml. of 2 M ammonium chloride was added.

TABLE

II

DISTRIBUTION OF L-LEUCINE AND ACID DEHYDROGENASE ACTIVITIES CENTRIFUGATION

L-GLUTAMIC AFTER

Initial protein concentration and conditions of centrifugation are described in the text. Protein recovery values indicate the percentage of the protein original solution which was recovered in the supernatant fluid or the pellet -

Expk

Fraction

I

T

(2) L-

Glutamate __--

2 ;: 5 .,o 2

hr.

1 2

Control Supernatant Pellet Supernatant Pellet

-

1

1 2 2

1.62 0.87 0.73 0.37 1.48

90.1

43.0 41.5 19.5 73.5

/ 56

50 57 53 50

22 80 53 44

7.4, and incubated at 50°C. At various time intervals, 0.20 ml. of the solution was pipetted into 1.80 ml. of ice-cold buffer solution. The samples were then centrifuged to remove denatured protein, and the supernatant solutions were assayed for activity toward L-leucine and L-glutamic acid. Figure 2 shows that during the 60-min. heating

264

STRUCK AND SIZER

bered 11, 12, and 13 contained a significant amount of protein. The total amount in these three eluates accounted for 78% of that applied. Apparently the remainder of the protein was irreversibly adsorbed to the paper. Figure 3 indicates a close correlation between protein concentration and enzymic activity toward both leucine and glutamic acid. TIME AT 50°, MIN FIG. 2. Heat inactivation of crystalline glutamic acid dehydrogenase. Residual activity is plotted as a function of incubation time at 50°C. Conditions of experiment as described in text.

20

5

0 6

8

IO 12 14 TUBE NUMBER

16

I8

0

FIG. 3. Paper electrophoresis of crystalline glutamic acid dehydrogenase. Separation of enzyme fractions by continuous electrophoresis using a paper curtain. Conditions of electrophoresis as described in text. Protein was determined by absorbance at 279 mLc.

GLUTAMIC ACID DEHYDROGENASE PH-ACTIVITY CURVES FOR GLUTAMIC ACID AND LEUCINE

The activity of crystalline glutamic acid dehydrogenase toward L-glutamic acid and r,-leucine was examined as a function of pH (Fig. 4). Activities were estimated from the rate of DPNH formation under the conditions described for enzyme assay. In these determinations the final buffer concentration was 0.1 M and the pH of the reaction mixture was measured immediately after assay. During the study of the effect of pH on the rate of reaction, it was observed that above pH 8.0 the rate of DPNH formation decreased rapidly with time. This decrease was such that the estimation of initial velocity was unsatisfactory above pH 8.0. It was found that this difficulty could be overcome by stabilizing the enzyme with I

I

I

I

I

I

-

IO: 5 a-c 2 6-

period the loss of activity toward both substrates occurred at identical rates. An additional attempt was made to separate the two activities by subjecting a samplk of crystalline glutamic acid dehydrogenase to electrophoresis in a Beckman (model CP) continuous-flow paper electrophoresis cell at 60 ma. Ten milliliters of a solution of the enzyme in 0.04 M potassium phosphate buffer, pH 7.0, was subjected to electrophoresis at 2°C. using a curtain irrigated with the same buffer. After completion of the run, protein concentration was estimated in each of the 32 eluate tubes from the absorbance at 279 mp. Only tubes num-

Y c 4< ~ii 20

6

GLUTAMIC ACID qi\l 7

8

LEUCINE 9

8

9

lo

PH FIG. 4. The rates of oxidation by glutamic acid dehydrogenase of L-leucine and L-glutamic acid as a function of pH. The rate of DPNH formation was determined spectrophotometrically at 340 mp. Reaction mixtures contained 300 pmoles buffer, 50 @moles substrate, 3 pmoles DPN, 1.5 mg. plasma albumin, and enzyme in a total volume of 3.0 ml. The buffers used were Tris (0) and glyoine (Cl).

265

GLUTAMIC ACID DEHYDROGENASE

0.05 % plasma albumin. The presence of albumin was found to have no effect on the rate of reaction over the 4-min. assay period.

TABLE

III

OXIDATION OF AMINO ACIDS BY CRYSTALLINE GLUTAMIC ACID DEHYDROGENASE

Conditions of assay are described in the text. SUBSTRATE SPECIFICITY OF CRYSTALLINE GLUTAMIC ACID DEHYDROGENASE

Following the demonstration that L-leutine could serve as the substrate for crystalline glutamic acid dehydrogenase, the specificity of this enzyme was investigated. The results obtained with various substrates are recorded in Table III. Rates of oxidation as indicated by DPNH formation are expressed relative to the rate of oxidation of glutamic acid in 0.1 M Tris, pH 8.0. The rates of oxidation of other amino acids were measured in 0.1 M Tris, pH 9.0. Where a relative rate of ISCUSSION

The fact that the ratio of oxidation rates of n-leucine and L-glutamic acid is the same for the chicken liver mitochondrial enzyme in several stages of purity and is identical for crystalline glutamic dehydrogenase of beef liver suggests that a single enzyme is involved. This conclusion is further supported by the fact that this ratio is unaffected by treatment of the crystalline enzyme in a variety of ways. Failure of previous workers to detect the oxidation of other amino acids by glutamic dehydrogenase may be partially explained by the wide difference between the rates of oxidation of glutamic acid and other substrates. Part of the explanation lies in the different pH dependence of the oxidation of n-glutamic acid and Lleucine. Whereas the optimum pH for the oxidation of glutamic acid occurs at approximately 8.4 in Tris buffer, the oxidation of L-leucine (optimum pH about 10) at pH 8.4 is only about 0.5 % of that of L-glutamic acid. It now appears that this difference in pH optimum cannot be explained on the assumption that two different enzymes are involved, but that it is related to the difference between dicarboxylic and monocarboxylic acid substrates. The data recorded in Table III show that

Substrate

n-Glutamic acid n-Norvaline n-a-Aminobutyric acid L-Leucine L-Valine on-Norleucine L-Isoleucine L-Methionine n-Alanine L-Ornithine, n-lysine, n-proline, L-aspartic acid, r.-cu-aminoadipic acid, L-threonine, n-leucine, and nn-a-methylglutamic acid, (all)

Relative rate

1CKl 17 2.3 1.7 1.6 1.6 0.95 0.82 0.27


at least eight amino acids in addition to glutamic acid are oxidized by the crystalline dehydrogenase. Structural features which appear to govern the rate of oxidation are the L configuration of the a-amino group, length of the carbon chain, and the presence of the y-carboxyl group. In agreement with previous findings, this enzyme is found to exhibit a stereospecificity for the L isomer. Consideration of the dicarboxylic amino acids tested shows that the second carboxyl group must be in the y-position with respect to the amino group if the reaction is to occur. A decrease or increase of the length of the carbon chain by a single methylene group (e.g., aspartic and a-aminoadipic acids) appears to abolish activity. In the absence of the second carboxyl group the aliphatic amino acids undergo oxidation, although at a reduced rate. The fact that the diamino acids, ornithine and lysine, do not serve as substrates suggests that the y-carboxyl group of glutamic acid facilitates the binding of this substrate to the enzyme through an electrostatic attraction between this negatively charged group and a positively charged group on the enzyme. The presence of a second positively charged group in the substrate, as in ornithine or lysine, would interfere with binding to the enzyme. Since amino acids other than n-glutamic acid can serve as substrates for glutamic

266

STRUCK

acid dehydrogenase, this reaction may serve as a possible mechanism for their deamination in vivo, although the low rates of reaction would seem to preclude this as a major metabolic pathway. In this connection, it is interesting to note that a pyridine nucleotide-mediated oxidation of certain L-amino acids would serve as an explanation of Krebs’ (9) observation that the oxidation of L-amino acids by rat kidney slices was inhibited by cyanide and octyl alcohol, since these inhibitors would be effective in preventing the reoxidation of the reduced nucleotide. The reversibility of the glutamic acid dehydrogenase reaction provides a mechanism for the direct incorporation of ammonia into amino acids. It is possible that the nonspecific action of this enzyme may account for the direct synthesis of alanine from pyruvic acid and ammonia in homogenates of rat liver (10-12) and in bacterial extracts (13, 14). Although the mechanism of the in viva amination by ammonia of cr-keto acids remains controversial (15), the observation of the reversible oxidation-reduction of amino acids by glutamic acid dehydrogenase provides an enzymic basis for such a reaction, ACKNOWLEDGMENTS The authors wish to thank Drs. W. T. Jenkins and R. W. Hartley for assistance with the determination of the electrophoretic and sedimentation

AND

SIZER

patterns of crystalline glutamic genase. This work was supported the National Institutes of Health con, Incorporated.

acid dehydroby grants from and from Ethi-

REFERENCES 1. WIAME, J. M., AND PIERARD, A., Nature 176, 1073 (1955). 2. ADLER, E., HELLSTROM, V., GUNTHER, G., AND EULER, H. VON, 2. physiol. Chem. 266, 14 (1938). 3. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 197, 67 (1952); aO2, 84 (1953). 4. STRECKER, H. J., Arch. Biochem. Biophys. 33, 448 (1941); 46, 128 (1953). 5. ADICKES, F., AND ANDRESEN, G., Ann. 666, 41 (1943). 6. MOKRASCH, L. C., AND MCGILVERY, R. W., J. Biol. Chem. 221, 909 (1956). 7. BOYER, P. D., AND SEGAL, H. L., in “The Mechanism of Enzyme Action” (McElroy, W. D., and Glass, B., eds.). Johns Hopkins Press, Baltimore, 1954. 8. STRUCK, J., AND SIZER, I. W., Federation Proc. 16, 257 (1957). 9. KREBS, H. A., Biochem. J. 29, 1620 (1935). 10. WISS, O., Helv. Chim. Acta 31, 1189 (1948). 11. KAPLANASKY, S. Y., AND BEREZOVSKAYA, N. N., Biokhimiya 21, 119 (1956). 21, 733 12. BEREZOVSKAYA, N. N., Biokhimiya (1956). 13. FAIRHURST, A. S., KING, H. K., AND SEWELL, C. E., J. Gen. Microbial. 16, 106 (1956). 14. GOWLER, E. B., AND WERKMAN, C. H., Arch. Biochem. Biophys. 41, 42 (1952). 15. BRAUNSTEIN, A. E., Advances in Enzymol. 19, 8.15 (1957).