Rat Liver Aminomalonate Decarboxylase

THE JOURNAL OA Bmmorca~ CHEMISTRY Vol. 248, No. 4,Issue of February 25, pp. 1158-1167, 1973

Petea in U.S.A.

Rat Liver Aminomalonate Decarboxylase

IDENTITY WITH CYTOPLASMIC SERINE HYDROXYMETHYLASE AND ALLOTHREONINE ALDOL- ASE*

(Received for publication, October 6, 1972)

ANIL G.PALEKAR,SURESH S.TATE, AND ALTON MEISTER

From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021

SUMMARY

Studies on the aminomalonate decarboxylase of rat liver indicate that this activity is a property of cytoplasmic serine hydroxymethylase. Thus, throughout purification of the enzyme, aminomalonate decarboxylase, serine hydroxymethylase and allothreonine aldolase exhibited the same relative activities. Competition between the several substrates was observed and resolution of the enzyme by treatment with D-alanine led to loss of all three activities. Cleav- age of allothreonine by the enzyme in tritiated water gave S-glycine-2-t (in accord with earlier data which indicate retention of configuration during conversion of L-serine to glycine), but decarboxylation of aminomalonate by the enzyme in tritiated water gave both S- and R-glycine-2-t. Studies with specifically carboxyl-labeled [14C]aminomalonate confirmed the conclusion that the enzyme decarboxylates this amino acid in a nonspecific manner; this result is in contrast to that previously observed with aspartate ,&decarboxylase which acts on a specific carboxyl group of aminomalonate. In contrast to cytoplasmic serine hydroxymethylase, mitochondrial serine hydroxymethylase does not catalyze the aldol cleavage of allothreonine or the decarboxylation of aminomalonate; this indicates that there is a significant difference between the active sites of these enzymes.

Aminomalonic acid, an amino acid which has not yet been found in nature, has nevertheless been of interest to biochemists because (a) of the possibility that it might be an intermediate in the conversion of serine to glycine (consideration of this symmetrical compound as a possible intermediate led to the Ogston hypothesis (1)) ; (b) enzyme activity capable of decarboxylating aminomalonic acid to glycine has been found in several biological materials; and (c) aminomalonic acid, a lower homolog of as- partic acid, inhibits certain enzyme-catalyzed reactions involving this and other amino acids.

The present studies on the purification and properties of rat liver aminomalonate decarboxylase activity were stimulated by the observation (2, 3) that bacterial L-aspartate P-decarboxylase catalyzes the specific removal of the carboxyl group of aminomalonic acid that is in the position analogous to the -CH&OO-

* This research was supported in part by the National Institutes of Health, United St,ates Public Health Service.

group of L-aspartate rather than to the oc-carboxyl group. It had been shown earlier that preparations of the posterior silk glands of silk worms (4) and of rat liver (5) contain enzyme activity capable of decarboxylating aminomalonic acid to glycine. We sought to determine whether the decarboxylation of aminomalonic acid catalyzed by liver exhibits a specificity similar to or different from that catalyzed by L-aspartate fi-decarboxylase. In the present investigation we have found that the decarboxylation of aminomalonic acid by the activity present in rat liver does not exhibit specificity toward a single carboxyl group. In the course of this work, we have obtained evidence that the aminomalonate decarboxylase activity exhibited by rat liver preparations is associated with soluble serine hydroxymethylase. It is of interest that the aminomalonate decarboxylase activity exhibited by this enzyme is several times greater than the serine hydroxymethylase activity, and that the decarboxylase activity does not require tetrahydrofolic acid. It was also found in the present work that serine hydroxymethylase prepared from rat liver mitochondria does not exhibit aminomalonate decarboxylase activity, thus indicating an interesting difference between the soluble and particulate forms of serine hydroxymethylase.

EXPERIMENTAL PROCEDURE

Materials

Unlabeled and [2-14C]- and [3-‘%]aminomalonic acid were synthesized as described previously (2, 3, 6). L-Threonine and m-allothreonine were obtained from Calbiochem. Pyridoxal 5’-phosphate, DPNH, TPN, and tetrahydrofolic acid were obtained from Sigma Chemical Co. Yeast alcohol dehydrogenase was obtained from Worthington Biochemical Corp. The analogs of pyridoxal %phosphate were obtained as noted previously

(7). L-Allothreonine was prepared by the method of Elliott (8) as described by Greenstein and Winitz (9). The N-oxide of pyridoxal 5’-phosphate was kindly donated by Dr. S. Fukui, Kyoto, Japan. We are indebted to Dr. Daniel Wellner of this department for a generous supply of hog kidney D-amino acid oxidase.

Methods

Determination of Aminomalonic Decarboxylaae Activity

The assay mixtures (final volume, 0.1 ml) contained potassium phosphate buffer (6 pmoles; pH 6.0), [2-14C]aminomalonate (2.5 pmoles; 40,000 cpm per pmole), pyridoxal 5’-phosphate

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(1 nmole), dithiothreitol (10 nmoles), and enzyme. After incubation for 30 min at 37”, aliquots (50 ~1) were withdrawn and applied to small columns of Dowex 1 X-8 (acetate form) prepared in Pasteur pipettes. The radioactive glycine formed in the reaction was eluted with 1 ml of water and the eluate was mixed with 10 ml of liquid scintillation medium (10) ; radioactivity was determined in a liquid scintillation counter. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 nmole of glycine per min at 37”.

Determination of L-Se&e Hydroxymethylase Activity

Assay I-This activity was determined essentially as described by Schirch (11) in reaction mixtures containing 5, IO-methylene tetrahydrofolate dehydrogenase (prepared from chicken liver (12)) and TPN. The formation of TPNH was determined from the increase in absorbance at 340 nm. The assay mixture (final volume, 1 ml) contained potassium phosphate buffer (100 pmoles; pH 7.5), dl-n-tetrahydrofolate (0.25 pmole), dithiothreitol (1 pmole), pyridoxal 5’-phosphate (10 nmoles), TPN (0.4 pmole), n-serine (5 pmoles), 5, IO-methylene tetrahydrofolate dehydrogenase, and serine hydroxymethylase. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 nmole of TPNH per min at 37” under these conditions.

Assay .%-This activity was also determined by measuring the rate of disappearance of formaldehyde in the presence of glycine (13). The assay mixtures (final volume, 0.1 ml) contained in addition to the enzyme, potassium phosphate buffer (10 pmoles; pH 7.5), dl-n-tetrahydrofolate (0.1 Imole), pyridoxal 5’-phosphate (1 nmole), formaldehyde (0.5 pmole), dithiothreitol (50 nmoles), and glycine (5 pmoles). After incubation at 37” for 10 min the reaction was stopped by adding 0.1 ml of 10% tri- chloroacetic acid. Controls lacking enzyme were carried out. The remaining formaldehyde was determined by a modification of methods previously described for the determination of bisulfite (14, 15). Aliquots (0.1 ml) were made to 4.5 ml with water and then treated with 0.5 ml of a 1: 1 (v/v) mixture of 0.01 M NaHS03 and 0.047, p-rosaniline HCl. The solutions were mixed and incubated at 37” for 45 min; the colors were then compared at 560 nm. Under these conditions, 0.1 pmole of formaldehyde gave an absorbance of 0.5. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the disappearance of 1 nmole of formaldehyde per min under these conditions. The results obtained by this assay procedure were, within experimental error, the same as those obtained by Assay 1. Assay 2 was more convenient in experiments requiring a large number of analyses.

Determination of L-Allothreonine (and z-Threonine) Aldolase Activity

The method employed is a modification of that of Malkin and Greenberg (16). The reaction mixture (final volume, 1 ml) contained potassium phosphate buffer (100 pmoles; pH 7.3), nn-allothreonine (10 pmoles), or n-threonine (40 pmoles), pyridoxal 5’-phosphate (10 nmoles), dithiothreitol (1 pmole), DPNH (150 nmoles), yeast alcohol dehydrogenase (35 units), and the enzyme. The assay mixture was equilibrated at 37” in a water jacketed cuvette compartment of a Cary model 15 spectrophotometer, and the reaction was initiated by adding the enzyme. The decrease in absorbance at 340 nm was recorded. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 nmole of acetaldehyde per min.

Protein was determined as described by Lowry et al. (17). Specific enzymatic activities are given in terms of units per mg of protein.

RESULTS

Initial Studies on Purification of Rat Liver Aminomalonate Decarboxylase Activity

Thanassi and Fruton (5) first observed the aminomalonate decarboxylase of rat liver and obtained about a 2-fold purification of this activity from rat liver acetone powder. In the present work we pursued the purification of aminomalonate decarboxylase activity from rat liver acetone powder and obtained, after chromatography on hydroxylapatite and DEAE-cellulose and gel filtration, preparations of relatively high specific activity, i.e. in the range of 10,000 units per mg. Since there seems to be no evidence indicating that aminomalonate is a metabolite, we considered the possibility that the purified enzyme preparations obtained might contain another pyridoxal 5’-phosphate enzymatic activity. The highly purified aminomalonate decarboxylase preparations were therefore tested for the following enzyme activities: n-serine dehydratase, n-allothreonine aldolase, n-threonine aldolase, n-threonine dehydratase, n-serine hydroxymethylase, n-cysteine desulfhydrase, cystathionase, n-aspartate fl-decarboxylase, n-aspartate a+decarboxylase, n-cysteine sulfinate decarboxylase, kynureninase, sphingosine synthetase, alanine racemase, glutamate-aspartate transaminase, and glutamate-alanine transaminase. The preparation was also tested for ability to catalyze transamination between oc-ketoglutarate and n-leucine, n-serine, n-threonine, and glycine, and between glyoxylate and aminomalonate, n-asparagine, n-glutamine, n-aspartate, and n-alanine. The only catalytic activities detected were serine hydroxymethylase, allothreonine aldolase, and threonine aldolase. In addition, when the various fractions obtained during purification were examined for serine hydroxymethylase and allothreonine aldolase activities, it was found that these activities had been purified to about the same extent as aminomalonate decarboxylase. A number of such studies were carried out on several highly purified aminomalonate decarboxylase preparations with similar results. We then decided to use a method of purification that is substantially the same as that described by Nakano et al. (18) for the isolation of cytoplasmic rat liver serine hydroxymethylase. In the present studies, the procedure of Nakano et al. (18) was modified in certain respects; thus, dithiothreitol was included in all of the buffers used. In our hands, dithiothreitol was found to yield a more stable enzyme preparation. In addition, a new step involving chromatography on Sephadex G-150 was included; this facilitated the isolation of an apparently homogeneous enzyme as described below.

PuriJication of Aminomalonate Decarboxylase of Rat Liver and Its Apparent Identity with Cytoplasmic

L-Serine Hydroxymethylase and L-Allothreonine Aldolase

Male Sprague-Dawley rats, weighing approximately 300 g, were fasted for 24 hours prior to death by decapitation and removal of the livers. All subsequent steps were carried out at 4”.

Step I-The livers (450 g) were minced and homogenized in a Potter-Elvehjem homogenizer with 900 ml of 0.25 M sucrose containing 5 mM potassium phosphate buffer (pH 7.2) and 1 mM EDTA (sucrose-phosphate buffer) ; 2250 ml of sucrose-phosphate


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buffer were added to this homogenate and the mixture was stirred adding solid ammonium sulfate. The precipitate which formed for 15 min. The solution was then centrifuged at 15,000 x g was collected by centrifugation and dissolved in 1 ml of 0.05 M for 30 min. Assays for aminomalonate decarboxylase activity potassium phosphate buffer (pH 6.5). This solution was applied showed that more than 90% of the activity of the homogenate to a Sephadex G-150 column (3 x 55 cm), and elution was car- was present in the supernatant solution after centrifugation. ried out with the same buffer. The fractions (2.2 ml each) ex- (The pellet was used for the preparation of mitochondrial L-ser- hibiting high enzyme activity (Numbers 34 to 40) were pooled ine hydroxymethylase; see below.) (Table I, Fig. 1).

Step g-The supernatant solution obtained in Step 1 was brought to 43oj, of ammonium sulfate saturation by adding solid ammonium sulfate; the suspension was then centrifuged at 15,000 X g for 20 min. The precipitate was discarded and the supernatant solution was brought to 537, of ammonium sulfate saturation by adding solid ammonium sulfate. The precipitated protein was centrifuged, dissolved in 300 ml of 0.02 M potassium phosphate buffer (pH 7.2) containing 1 mM EDTA, 0.2 mM dithiothreitol and 10e5 M pyridoxal 5’.phosphate, and then this solution was throughly dialyzed against the same buffer. All of the buffers used in the subsequent steps of purification contained 1 MM EDTA, 0.2 mM dithiothreitol and lo+ M pyridoxal 5’-phosphate.

Step r--Sufficient L-serine was added to the pooled enzyme solution obtained in Step 6 to give a final concentration of 1 mM. This solution was applied to a DEAE-cellulose column (2.5 X 10 cm) which had been equilibrated with 0.05 M potassium phos-

Step S-The dialyzed solution obtained in Step 2 was diluted with 0.02 M potassium phosphate buffer to give a protein concentration of 20 mg per ml. To this solution were added, 0.2 volume of 0.5 M potassium phosphate buffer (pH 6.5) and solid L-serine (final concentration, 10 mM). The solution was then brought to 70” and kept at this temperature for 3 min; it was then rapidly cooled and the denatured protein was removed by centrifugation.

Step C-Solid ammonium sulfate was added to the supernatant solution obtained in Step 3 to obtain 53% of ammonium sulfate saturation. The precipitate which formed was dissolved in 30 ml of 0.05 M potassium phosphate buffer (pH 6.5), and this solution was dialyzed against 100 volumes of the same buffer.

Step 5-The dialyzate obtained in the preceding step was applied to the top of a DEAE-cellulose column (Whatman DE-52 microgranular, preswollen; 3.2 x 10.8 cm), which had been equilibrated with 0.05 M potassium phosphate buffer (pH 6.5). The column was washed with the same buffer until protein no longer emerged. Further elution was then carried out with a linear gradient established between 1 liter each of 0.05 Y and 0.25 M potassium phosphate buffer (pH 6.5). The fractions exhibiting high enzyme activity were pooled.

45 50 55

FRACTIONS

Step 6-The pooled enzyme solution obtained in the previous step was brought to 53% of ammonium sulfate saturation by

FIG. 1. A, chromatography of the enzyme on Sephadex G-150 (Step 6 of the purification procedure; see the text). B, chromatography of the enzyme on DEAE-cellulose (Step 7 of the purification procedure; see the text).

TABLE I

Szcmmary of purijication of rat liver aminomalonate decarboxylase

Purification step

1. Rat liver extract (soluble fraction). 2. Ammonium sulfate fractionation.. 3. Heat denaturation of impurities. -1. Ammonium sulfate fractionation. 5. DEAE-cellulose chromatography.. 6. Sephadex G-150 chromatography.. 7. DEAE-cellulose chromatography..

-

I. Volume

COlICen- tration

Vd mvml 3,110 21.0

350 34.6 620 3.16

34 26.1 72 0.21 17.5 0.40 2.6 0.58

Protein

Total mg Total units

65,300 643,000 12,100 490,000

1,960 440,000 887 364,000

15.1 115,000 7.0 92,500 1.5 23,200

Activity= I

Ratio of specific activities I

I I

Amino- malonate

Specific

unifs/mg

9.85 2.70 40.5 3.14

225 3.21 410 3.10

7,620 3.10 13,200 3.08 15,500 3.04

-

) I

L-Allo- threonine aldolase

to I- threonine aldolase

Amino- malonate decarboxylase to L-wine hydroxymethylase

2.68 3.34 3.38 3.14 3.10 3.16 3.07

Yield

___~ %

3 100 5 76 7 68

13 57 21 18 50 14 m 4

D Enzyme activity was measured as described in the text.


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phate buffer (pH 6.5) containing 1 mM L-serine. The column was washed with the same buffer until protein no longer emerged. Further elution was carried out with a linear gradient established between 400 ml each of 0.05 M and 0.2 M potassium phosphate buffer (pH 6.5) containing 1 mM n-serine. Fractions of 6 ml were collected. Fractions 46 to 49 (Fig. IL?), which exhibited the highest specific activity, were concentrated by ultrafiltration. The enzyme preparation exhibited aminomalonate decarboxylase, n-serine hydroxymethylase, and n-allothreonine aldolase activities with respective specific activities of 15,000, 5,050 and 5,100. The aldolase activity was determined with nn-allothreonine and with L-allothreonine; the rates of aldol cleavage were the same with nonsaturating concentrations of nn-allothreonine that were twice that of n-allothreonine, indicating that the enzyme is probably active only towards n-allothreonine. Addition of tetrahydrofolate did not affect the aminomalonate decarboxylase or allothreonine aldolase activities. A summary of the purification procedure is given in Table I.

As stated above, the aminomalonate decarboxylase activity was found only in fractions which also contained n-serine hydroxymethylase and n-allothreonine aldolase activities. Fur- thermore, the ratios of these activities remained constant throughout purification (Table I) ; thus, the ratios of decarboxylase to hydroxymethylase and of decarboxylase to aldolase were close to three for all fractions. The elution patterns shown in Fig. 1 also indicate that the three activities emerge together from the Sephadex and DEAE-cellulose columns.

Further evidence that the three activities are associated with the same protein was obtained from the finding of a single protein band on polyacrylamide gel electrophoresis at pH values of 7.5, 8.0, and 8.5. The electrophoreses were performed in 5% polyacrylamide gels according to the procedure of Davis (19) ; the buffers used were 0.05 M potassium phosphate (pH 7.5), and 0.05 M Tris-acetate (pH 8.0 and 8.5). Fig. 2B shows the pat- tern obtained at pH 8 with the fraction obtained from the second DEAE-cellulose column (Step 7, Table I) ; Fig. 2A shows that more than one protein is present in the eluate from the Sephadex G-150 column (Step 6, Table I).

Gel electrophoresis was also performed according to Weber and Osborn (20) on 6% and 7.5% gels in a continuous buffer system consisting of 0.05 M Tris-acetate (pH 8.5) containing 0.1% (w/v) sodium dodecyl sulfate. The gels were stained with 0.25% Coomassie blue in methanol-acetic acid-water (5: 7.5 : 87.5, v/v). Chymotrypsinogen, ovalbumin monomer and

__’

A g : a- %a 4

FIG. 2. Polyacrylamide gel electrophoresis of aminomalonate decarboxvlase (n-serine hvdroxvmethvlase). Electronhoresis was carried out on 5% gels prepared in 0.65 M Tris-acetate buffer (pH 8.0). A. the eluate from Senhadex G-150 @ten 6. Table I): 20 pg of protein. The gel was r;n for 2 hours; 5 ma per gel. Bl’the eluate from the second DEAE-cellulose column (Step 7, Table I) ; 15 rg of protein. The gel was run for 3 hours.

dimer, bovine serum albumin (monomer, dimer, trimer), and n-asparate /3-decarboxylase monomer (21) were used as standards. A plot of the molecular weights of the marker proteins against their mobilities is shown in Fig. 3. Aminomalonate decarboxylase (serine hydroxymethylase) gave a single discrete band on such gels yielding a monomer molecular weight of 61,000 f 2,000.

It is notable that the ratio of L-allothreonine aldolase activity to n-threonine aldolase activity increased considerably during purification (Table I). Indeed, the final preparation exhibited no activity towards n-threonine. This seems to indicate that the cleavage of n-threonine is probably catalyzed by a different enzyme.

Since catalysis of the interconversion of n-serine and glycine is the only known physiological function of the enzyme isolated in the present studies, we refer to it as serine hydroxymethylase in the following text.

Studies on Mitochondrial &S’erine Hydroxymethylase

Mitochondrial n-serine hydroxymethylase was purified as described by Nakano et al. (18) except that chromatography on brushite was omitted. Aminomalonate decarboxylase, L-allothreonine aldolase, and threonine aldolase activities were not detected in the mitochondrial extract, the initial fractions, or a preparation purified by passage through DEAE-cellulose. The mitochondrial enzyme therefore differs markedly in its substrate specificity from the cytoplasmic enzyme. We also found that the mitochondrial enzyme exhibits a substantially lower mobility than does the cytoplasmic enzyme on polyacrylamide gel electrophoresis; thus, the two enzymes were completely separated under the conditions given in Fig. 2B.

T WA-3

11 15 30 45 60 75 90

MIGRATION, mm

FIG. 3. Molecular weight calibration curve ‘for 6% (Curve 1) and 7.5% (Cztrve .%) polyacrylamide gels run in 0.1% sodium dodecvl sulfate. The protein standards were: chgmotrgpsinogen (CH);Vovalbumin monomer and dimer (OV-1 and OV-i); bo&e serum albumin (BSA-1, -2, -5) ; and L-aspartate p-decarboxylase from Alcali~enes faecalis (ASPD; mol wt of monomer, 57,000). Prior to ele&rophoresis the proteins were incubated for 3 hours at 25” in Tris-acetate buffer (nH 8.5) containing 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. The latter-was omitted in the studies on ovalbumin and serum albumin. AMAD, aminomalonate decarboxylase (n-serine hydroxymethylase).


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Properties of Cytoplasmic Serine Hydroxymethylase

The effect of varying pH on the three activities catalyzed by cytoplasmic serine hydroxymethylase is described in Fig. 4. The aminomalonate decarboxylase activity was maximal at values of pH about 5.5 to 6.0; studies were not carried out at lower values of pH at which the substrate undergoes spontaneous decarboxylation. Allothreonine aldolase activity exhibited a broad pH optimum between about pH 6.5 and 7.5. Serine hydroxymethylase activity was maximal between pH 7.5 and 8.0. Nakano et al. (18) reported a pH optimum of 8.0 for cytoplasmic serine hydroxymethylase; the optimal pH for both cytoplasmic and mitochondrial rabbit liver serine hydroxymethylases has been reported to be pH 7.5 (22).

The absorption spectrum of the purified cytoplasmic serine hydroxymethylase exhibited maxima at 280 and 430 nm (Fig. 5, Curve 1). Determination of the pyridoxal 5’-phosphate content of the isolated holoenzyme (after dialysis against 0.05 M potassium phosphate buffer (pH 6.5) containing 0.1 mM dithiothreitol) by the phenylhydrazone method of Wada and Snell (23) gave a value of 1 mole of pyridoxal5’-phosphate per 59,000 g of protein. Treatment of the enzyme with sodium borohydride led to disappearance of the maximum at 430 nm and the appearance of a new peak at 330 nm (Fig. 5, Curve 2) ; the reduced enzyme exhibited none of the enzyme activities.

Schirch and Jenkins (24) reported that serine hydroxymethylase catalyzes a relatively slow transamination reaction between n-alanine and enzyme-bound pyridoxal 5’-phosphate to yield pyruvate and the pyridoxamine %phosphate form of the enzyme; this enzyme form exhibits no serine hydroxymethylase activity. In the present studies, the purified enzyme was

I i r-----l”’

incubated with n-alanine and the transamination reaction was followed by measuring the disappearance of the absorbance maximum at 430 nm; simultaneous determinations of serine hydroxymethylase and aminomalonate decarboxylase activities were performed. As indicated in Fig. 6, the decrease in absorbance at 430 nm was associated with simultaneous de- creases in the serine hydroxymethylase and aminomalonate decarboxylase activities. However, when these activities were determined in the presence of added pyridoxal p-phosphate restoration of both activities was observed.

The purified serine hydroxymethylase was resolved from its vitamin Bs cofactor by incubation of the enzyme with n-alanine for 2 hours at 37”; the solution was then dialyzed against 0.05 M

potassium phosphate buffer (pH 6.5) for 18 hours. The apoenzyme solution exhibited no characteristic absorbance at either 430 or 330 nm, nor did it have any of the catalytic activities exhibited by the holenzyme. When the apoenzyme was incubated with pyridoxal Y-phosphate, each of the three enzyme activities was restored. The apoenzyme was also incubated with a variety of pyridoxal phosphate analogs as indicated in Table II. Relatively similar activity values were obtained with 2-norpyridoxal 5’-phosphate and pyridoxal F-phosphate N-oxide; however, the activities observed with the other pyridoxal analogs, while in most cases similar for serine hydroxymethylase and allothreonine aldolase, were substantially lower for aminomalonate decarboxylase. These findings may be a reflection of the fact that the enzyme exhibits a lower affinity for aminomalonate than for the other substrates (see below). It seems relevant to note that somewhat similar results have been obtained in studies on the effect of various vitamin Bg phosphate analogs on the activities catalyzed by asparate P-decar-

0 1 6.0 7.0 8.0 5:

PH

FIG. 4 (left). pH dependence of the enzyme activities. For the assay of aminomalonate decarboxylaee activity ( l -- l ) , the reaction mixtures contained 0.062 M potassium phosphate buffer (pH, 5.5 to 7.5), 25 mM [2-‘%]aminomalonate (40,000 cpm per pmole), 10e4 M dithiothreitol, 10e5 M pyridoxal 5’-phosphate, and the enzyme (2 pg). For the itssay of L-allothreonine aldolase (O-O), the reaction mixtures contained 0.1 M potassium phosphate buffer (pH 6.0 to 8.0), 10 mrvr nn-allothreonine, 10m5 M pyridoxal 5’.phosphate, 1 mM dithiothreitol, 1.5 X 10e4 M DPNH, yeast alcohol dehydrogenase (35 units), and enzyme (2pg). n-Ser- ine hydroxymethylase (A--A) was assayed in reaction mixtures containing 0.1 M potassium phosphate buffer (pH, 7.0 to 9.0), lop5 M pyridoxal 5’-phosphate, 1 m&f dZ,n-tetrahydrofolate, 5 rnM formaldehyde, 5 X 10d4 M dithiothreitol, 50 mM glycine, and the enzyme (2 rg). Other details are described under “Methods.”

FIG. 5 (center). Absorption spectra of the n-serine hydroxy-

TIME, MIN.

methylase. Curve 1, holoenzyme; Curve 2, holoenzyme after re- duction by sodium borohydride. Enzyme concentration was 0.8 mg per ml in 0.05 M potassium phosphate buffer (pH 6.5).

FIG. 6 (right). Disappearance of enzymatic activities and absorbance at 430 nm during incubation of the enzyme with n-alanine. The enzyme (0.35 mg; per 0.5 ml) was dialyzed against two changes each of 1000 volumes of 0.05 M potassium phosphate buffer (pH 6.5), containing 1 mM EDTA and 0.2 mM dithiothreitol. So- lid n-alanine was then added to give a final concentration of 0.2 M, and the decrease in absorbance at 430 nm was recorded at 37” with a Cary model 15 spectrophotometer. Aliquots were withdrawn for determinations of aminomalonate decarboxylase and n-serine hydroxymethylase activities. These assays were determined as described in the text in the absence (0, 0) and in the presence (A, A) of 10e5 M pyridoxal 5’-phosphate.


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boxylase. Thus, the relative decarboxylase, cysteine sulfinate desulfinase, and transaminase activities exhibited by this enzyme vary considerably depending upon the pyridoxal Sphos- phate analog attached to the enzyme (7). It is also notable that the optical specificity of aspartate /Sdecarboxylase can be substantially altered in this way; thus, the N-methylpyridoxal 5’-phosphate-enzyme catalyzes the transamination and the P-decarboxylation of D-aspartate, while the pyridoxal Sphos- phate form of the enzyme is L-specific (7).

The effect of L-allothreonine on aminomalonate decarboxylase activity and the effect of aminomalonate on L-allothreonine aldolase activity are described in Fig. 7. The data indicate that each substrate competitively inhibits the activity of the enzyme toward the other substrate. The Ki values obtained from these studies are similar to the respective K, values (Table III). Thus, the Kc value for L-allothreonine was 1.5 mM and the K, value for L-allothreonine as a substrate was 1.0 mM; sim- ilarly, the K, value for aminomalonate as a substrate (12.8 mM) is close to that for aminomalonate as an inhibitor of L-allothreonine aldolase activity (Ki, 10.0 mM). As indicated in Fig. 8,

TABLE II

Effect of vitamin B, phosphate analogs on enzyme activities

T I.

Cofactor or analog”

Pyridoxal 5’.phosphate. 100 100 100 2-Norpyridoxal 5’-phosphate. 53.4 54.5 55 2-Nor-2-ethylpyridoxal 5’.phosphate. 68.3 29.4 40 2-Nor-2-butylpyridoxal 5’-phosphate.. 1.9 0 1 3-O-Methylpyridoxal 5’-phosphate. . 8.1 0 7.5 AT-Methylpyridoxal 5’-phosphate. 4.7 0 5.1 Pyridoxal 5’-phosphate N-oxide. 90.1 81.5 88.7 Pyridoxal 5’-sulfate. 14.9 1 8.1

Relative activities

k%xine hydroxymeth-

ylase

Amino- L-Allo- malO- thre- nate mine

decar- aldo- boxylase lase

Q The apoenzyme (25 pg in 0.05 ml of 0.05 M potassium phosphate buffer, pH 6.5, containing low4 M dithiothreitol and 1 mn/r EDTA) was incubated with 5 nmoles of cofactor or analog for 30 min at 25” and then for 16 hours at 4”. The enzyme activities were determined as described under “Methods.”

d[AMINOMALONATE] x Kl-* 1/[DL-ALLOTHREONINE] x 10-3

FIG. 7. A, the effect of allothreonine on aminomalonate decarboxylase activity. The concentration of [2-14C]aminomalonate (40,000 cpm per pmole) was varied from 5 to 25 mM in the absence (Curve 1) and in the presence (Curve 2) of 5 mM nL-allothreonine. The reaction mixture contained 0.062 M potassium phosphate buffer (pH 6.3), lo-” M pyridoxal 5’-phosphate, 10W4 M dithiothreitol, and the enzyme (2 pg). B, the effect of aminomalonate on the allothreonine aldolase activity. The concentration of nL-allothreonine was varied from 0.5 to 5 mM in the absence (Curve 1) and in the presence (Cu2trve 2) of 10 mM aminomalonate; the reaction mixture also contained 0.1 M potassium phosphate buffer (pH 6.3), 10m5 M pyridoxal 5’.phosphate, 10W4 M dithiothreitol, 1.5 X lo-* M DPNH, yeast alcohol dehydrogenase (35 units), and the enzyme ,n \

L-serine is a competitive inhibitor of L-allothreonine aldolase activity; the Ki value (0.33 mM) is not far from that of the K,,,

value for L-serine (0.45 MM). These observations are in accord

with other data presented above, indicating that these three enzyme activities are catalyzed by the same enzyme protein.

The observation that L-serine hydroxymethylase can interact with D-alanine indicates that this enzyme exhibits some affinity for D-amino acids. Other studies have shown that D-serine and D-cysteine are competitive inhibitors of serine hydroxymethylase (25). Furthermore, as indicated in Table IV, both optical isomers of serine and cysteine inhibited the L-allothreonine aldolase

TABLE III

Apparent K, and Ki values T

Substrate PH Inhibitor JGfL

L-Serine. 7.5 L-Allothreonine. 7.3 L-Allothreonine. . 7.3 L-Allothreonine. 6.3 L-Allothreonine. 6.3 Aminomalonate. . . 6.0 Aminomalonate.. 6.3 Aminomalonate.. 6.3

nwl

0.45 0.25

L-Serine 1.0

Aminomalonate 12.0 12.8

L-Allothreonine

Ki

??zM

0.33

10.0

1.5

-1.0 1.0 2.0 3.0 4.0

t/[DL-ALLOTHREONINE] X lO-3

FIG. 8. The effect of L-serine on allothreonine aldolase activity. The concentration of nL-allothreonine was varied from 0.2 mM to 4 rnM in the absence (Curve f ) and in the presence (Cuurve 2) of 1 mM L-serine; the reaction mixture also contained 0.1 M potassium phosphate buffer (pH 7.3), 10e5 M pyridoxal 5’-phosphate, 10-4 M dithiothreitol, 1.5 X 1OW M DPNH, yeast alcohol dehydrogenase (35 units), and the enzyme (2 pg).

TABLE IV

Effect of optical isomers of serine and cysteine on L-allothreonine aldolase and aminomalonate decarboxylase activities

The enzyme activities were determined as described under “Methods” in the presence of inhibitor, 10 mM (allothreonine aldolase) and 25 mlw (aminomalonate decarboxylase).

Percentage of initial activity

Inhibitor Allothreonine Aminomalonate

aldolase decarboxylase

L-Serine .................. o-Serine .................. L-Cysteine ............... o-Cysteine. ..............

31 15 70 60 45 5 72 20


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and aminomalonate decarboxylase activities exhibited by the purified serine hydroxymethylase preparation.

Enzymatic Synthesis of Allothreonine

The enzyme-catalyzed condensation of glycine and acetaldehyde has been studied with liver preparations from rat (16) sheep (26, 27), and rabbit (28) ; the products formed were reported to be threonine (27), allothreonine (16), and both threonine and allothreonine (26). In the present studies when the purified serine hydroxymethylase preparation was incubated with labeled glycine and acetaldehyde, more than 97% of the product was found to be allothreonine. Thus, a reaction mixture (0.1 ml) containing 0.1 M potassium phosphat,e buffer (pH 7.3), 20 mM [lJ4C]glycine (180,000 cpm per pmole), 20 mM acetaldehyde, 10 j.6M pyridoxal 5’-phosphate, 0.1 mM dithiothreitol, and the enzyme (6 pg), was incubated at 37” for 30 min. The mixture was deproteinized by addition of 0.1 ml of ethanol and, after centrifugation, an aliquot of the supernatant solution was subjected to thin layer chromatography on Cellulose MN 300 (Eastman Kodak) precoated sheets in a solvent systeni consisting of t-butanol-methyl ethyl ketone-HnO-NHaOH (40 : 30 : 20 : 10, v/v) (26). The RF values in this system for glycine, allothreonine, and threonine are, respectively, 0.41, 0.58, and 0.73. The areas of the sheets corresponding to threonine and allothreonine were removed by scraping and the compounds were eluted with 2 ml of water. Aliquots of the eluates were taken for determinations of radioactivity. In this representative experiment, 222 and 5.5 nmoles of allothreonine and threonine, respectively, were formed.

Studies on Stereospecijicity of Aminomalonate Decarboxylase and L-Allothreonine

Aldolase Reactions

It has been shown that in the oxidation of glycine to glyoxylate by hog kidney n-amino acid oxidase, the hydrogen atom liberated in the course of oxidation is that which corresponds in position to the a-hydrogen atom of n-amino acids (29). Thus, n-amino acid oxidase is useful for the determination of the stereospecificity of reactions in which glycine is formed or utilized (2, 29-31). For example, when glycine-2-t was prepared either from L-serine in a reaction catalyzed by L-serine hydroxymethylase in tritiated water (29), or by decarboxylation of aminomalonate catalyzed by L-aspartate fi-decarboxylase in tritiated water (2), virtually all of the tritium in the glycine was released into water during its conversion to glyoxylate by n-amino acid oxidase; these findings led to the conclusion that the glycine-2-t prepared by these reactions is X-glycine-2-t. In a similar manner it was shown that R-glycine-2-t is produced by transamination in tritiated water between the pyridoxamine 5’-phosphate form of L-asparate @-decarboxylase and glyoxylate (2), or between L-asparate and glyoxylate in a reaction catalyzed by a rat liver transaminase (29).

In the present studies glycine-2-t was isolated from experiments in which the cleavage of L-allothreonine and the decarboxylation of aminomalonate by rat liver cytoplasmic hydroxymethylase were carried out in tritiated water (Table V). The samples of glycine-2-t thus obtained were then oxidized to glyoxylate with hog kidney n-amino acid oxidase. The data obtained indicate that virtually all of the tritium obtained in the oxidation of glycine-2-t derived by cleavage of allothreonine was released into water (Experiment 2, Table V) . On the other hand, only about half of the tritium in the glycine-2-t obtained by aminomalonate decarboxylation was released into water

TAHLE V

Zncorporation of tritium from H&t into glycine formed by decarboxylation of aminomalonate and by cleavage

of allothreonine Experiment 1: The reaction mixture (0.25 ml) cont,ained en-

zyme (20 pg), aminomalonate (7.5 pmoles), pyridoxal5’-phosphate (25 nmoles), dithiothreitol (250 nmoles), sodium phosphat,e buffer (pH 6.0; 15pmoles), and HzO-t (1.5 X lOlo cpm). After incubation at 37” for 30 min, the solution was applied to a column of Dowex 1 (acetate form). Glycine was eluted with 5 ml of water and the eluate was adjusted to pH 2.0 by adding acetic acid. It was then applied to a column of Dowex 50 (H+) which was washed with 10 ml

of water; the glycine was then eluted with 5 ml of 2 N NHaOH.

The eluate was lyophilized and the dried residue was dissolved in 0.4 ml of water; aliquots of this solution were analyzed for glycine

(32, 33) and radioactivity. Experiment 2: The reaction mixture (0.2 ml) contained enzyme

(40 pg), nL-allothreonine (15 pmoles), pyridoxal 5’-phosphate

(20 nmolesj, dithiothreitol (200 nmoles), potassium phosphate buffer (pH 7.3; 20 pmoles), DPNH (1 mg), yeast alcohol dehy-

drogenase (25 units), and HpO-t (3 X 1O’O cpm). After incubation

at 37” for 30 min, the pH of the solution was adjusted to 2 by

adding 2 N HCI, and it was then applied to a column of Dowex 50 (H+) which was washed with 10 ml of water. The glycine was then eluted (with allothreonine) with 5 ml of 2 N NH,OH. The

eluate was lyophilized and the dried residue was dissolved in 0.2 ml of water. Glycine was separated from allothreonine by

descending paper chromatography on Whatman No. 3MM paper

(pyridine-water, 80:20, v/v); under these conditions the RF values

for glycine and allothreonine were 0.25 and 0.50, respectively (28). The area corresponding to glycine was cut out and the amino acid

was eluted with the solvent; the solution was evaporated in vacua

to dryness and the residue was dissolved in 0.4 ml of water. Gly- tine was determined as in Experiment 1. Under the conditions used in Experiments 1 and 2 there was no detectable nonenzymatic

formation of glycine.

Experiment and procedure

1. Enzymatic decarboxylation of aminomalonate

2. Enzymatic cleavage of allothreonine

IlKOr- Gly-

Total pm- tine

dY- tion of

tine tritium “2:

formed into DOX gly- reac- tine tiona

pmoles CM pm& pmoles

3.00 90,000 1.6 2.93 85,GOO 1.1

1.6 92,000 0.4

H,O-t formed

Gly- OXYI- ate

formed Counts %

Per Tritium

min re- leasedb

/.moles

1.4 59,700 47.4 1.0 41,000 47.8

0.36 32,300 97.5

a The conditions used for the oxidation of glycine by n-amino- acid oxidase (DOX) were as follows. A sample (0.1 ml) containing glycine-t was treated with 0.2 ml of 0.1 M sodium pyrophos- phate buffer (pH 8.3), 0.15 ml of FAD (10 pg), 0.3 ml of a solution containing n-amino acid oxidase (1.5 mg) and bovine serum albumin (3 mg), and 10 ~1 of catalase (6900 units); the mixture was then incubated at 37” for 8 hours in a stoppered tube. The solution was then lyophilized and the distillate was quantitatively trapped in a Dry Ice-acetone bath. The volume of the condensate was measured and an aliquot was taken for determination of radioactivity. The lyophilized residue was dissolved in water and an aliquot was taken for determination of glyoxylate (34).

b Calculated from the amount of glycine oxidized to glyoxylate by n-amino acid oxidase.


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TABLE VI

Decarboxylation of [Z-~4C]aminomaZonate0

Experimentb

Enzymatic decarboxylation

[‘Cl- lUIIOk.5 Total ‘CO? Gly-

tine TotalC

CP% cPm cPm lzmoles

170 13,000 5,700 5,440 160

-1.

I -

Glycine

R&i- tive

specific activity

y Prepared from L-[3-‘4C]serine; see Palekar et al. (3). b The reaction mixture (0.2 ml) contained potassium phosphate

buffer (12 rmoles; pH 6.5), [3-‘4Claminomalonate (170 nmoles; 13,000 cpm), pyridoxal 5’-phosphate (2 nmoles), dithiothreitol (2 nmoles), and the enzyme (5 rg). After incubation for 30 min at 25”, the formation of ‘4COs and [14C]glycine was determined as described (3).

c Determined by the ninhydrin method (32).

(Experiment 1, Table V). As a control, unlabeled glycine (5 pmoles) was incubated with the hydroxymethylase in tritiated water under the same conditions used in this experiment. Gly- tine was then purified from the reaction mixture in the same way; the incorporation of tritium into glycine was only 246 cpm per pmole. A greater incorporation of tritium into glycine occurs at higher pH values (7.4, 7.5) and with higher concentrations of glycine (24, 30) ; such incorporation is increased about 500-fold when tetrahydrofolate is added (24).

Table VI summarizes the results of studies in which specifically labeled [3-14C]aminomalonate (i.e. prepared from L-[3J4C]serine (3)) was decarboxylated to glycine by serine hydroxymethylase. The findings indicate that the specific radioactivity of the [‘“Cl- glycine formed was close to 50% of that of the [3-14C]aminomalonate and that about 50% of the radioactivity was released as carbon dioxide. This shows that the enzyme does not catalyze the removal of a specific carboxyl group of aminomalonate.

DISCUSSION

The present studies indicate that the decarboxylation of aminomalonate (Reaction l), the aldol cleavage of L-allothreonine (Reaction 2), and the tetrahydrofolate-dependent interconversion of serine and glycine (Reaction 3), are catalyzed by the same enzyme protein of rat liver, i.e. cytoplasmic L-serine hydroxymethylase. The isolated enzyme, which is homogeneous by the criterion of polyacrylamide gel electrophoresis, exhibits these three activities in the same ratios as found for all of the partially purified fractions obtained.

Aminomalonate + glycine + CO2

z-Allothreonine e glycine + acetaldehyde

L-Serine + tetrahydrofolate = glycine

(1)

(2)

(3) + 5,10-methylene tetrahydrofolate

It seems probable that virtually all of the aminomalonate decarboxylase activity found in rat liver homogenates may be ascribed to cytoplasmic serine hydroxymethylase. This conclusion is supported by the studies described above, which include the effects on the several activities of resolution of the enzyme by reaction with n-alanine and the studies which indicate com-

1165

petition between the several substrates. The present and earlier studies on cytoplasmic serine hydroxymethylase thus indicate that this enzyme, like certain other vitamin B6 enzymes (3%39), is capable of catalyzing a number of chemical reactions. It is of interest that, while the hydroxymethylation reaction requires tetrahydrofolate, the aldol cleavage of allothreonine and the decarboxylation of aminomalonate do not; thus, the structure of the substrate appears to determine the reaction pathway.

There are apparently conflicting reports in the literature about the possible identity of serine hydroxymethylase, threonine aldolase, and allothreonine aldolase. Thus, Karasek and Green- berg (26) reported evidence for the presence of two distinct enzymes in sheep liver that catalyze, respectively, the cleavage of L-threonine and L-allothreonine, Malkin and Greenberg (16) reported evidence that a single enzyme in rat liver catalyzes the aldol cleavage of both L-threonine and L-allothreonine. The latter finding was contradicted by Riario-Sforza et al. (40), who presented evidence that threonine and allothreonine are cleaved by separate enzymes in rat liver. Schirch and Gross (28) have published evidence that a single enzyme in rabbit liver catalyzes serine-glycine interconversion, threonine cleavage, and allothreonine cleavage. While our results indicate that rat liver cytoplasmic serine hydroxymethylase catalyzes the aldol cleavage of L-allothrednine and the decarboxylation of aminomalonate, our most purified enzyme preparation exhibited no activity toward L-threonine. Indeed, the ratio of allothreonine aldolase activity to threonine aldolase activity increased considerably during purification, indicating that the cleavage of threonine is probably catalyzed by a separate enzyme; this is in accord with the conclusions of Riario-Sforza et al. (40). It is possible of course that the rat liver enzyme undergoes structural change during purification which alters the active site in such a way as to decrease its affinity for L-threonine. The contradictory reports in the literature concerning the cleavage of threonine and allothreonine may in part be influenced by the fact that some commercially available samples of threonine and allothreonine may be contaminated with each other. In addition, species differences apparently exist; thus, the ratio of serine hydroxymethylase to allothreonine aldolase activities for the rat liver enzyme studied here is close to one, while this ratio is about four for the rabbit liver enzyme (28). Furthermore, the reported specific activity (serine hydroxymethylase) of the rabbit liver enzyme is about twice that of the rat liver enzyme, and as stated above the rabbit liver enzyme was reported to catalyze the aldol cleavage of L-threonine, whereas our preparation from rat liver did not. It should also be noted that the monomer molecular weight of the rat liver serine hydroxymethylase as determined by sodium dodecyl sulfate gel electrophoresis is 61,000 f 2,000, while similar study of the rabbit liver enzyme gave a value of 53,000 f 5,000 (41).

A most interesting observation made in the present study is that rat liver mitochondrial serine hydroxymethylase purified by the method of Nakano et al. (18) exhibited no activity toward aminomalonate, allothreonine, or threonine. This striking difference between the specificities shown by the cytoplasmic and mitochondrial hydroxymethylases indicates that the struc- tures of the active sites of these enzymes must be quite different; indeed other differences between the two enzymes have been reported (18). It is conceivable that the mitochondrial serine hydroxymethylase is part of the mitochondrial glycine synthesis system, which catalyzes the formation of 2 moles of glycine from 1 mole each of serine, ammonia, and carbon dioxide (42-45).

There is substantial evidence that the serine hydroxymeth-


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EHZOH 3.

A H cCINH;

DOX H-C=Of3H20 H.C*NH$- 1 4.

T T T coo- coo- coo- coo-

5. L-SERINE AMINOMALONATE S-GLYCINE-2-t

6.

CH20H coo- A

NH;bC 4H NH;.:., r”“’ NH&H DOX , 3H-C=0 + Hz0

T T T coo- coo- coo-

. ;oo-

D-SERINE R-GLYCINE-2-t

FIG. 9. Formation of the S- and R-isomers of glycine-2-t from aminomalonate (see the text).

ylase reaction proceeds with retention of configuration about the cr-carbon atom of L-serine (29-31, 46, 47) ; when the reaction is carried out in tritiated water, S-glycine-2-t is formed since all of the tritium is released from glycine by n-amino acid oxidase (29). It may therefore be concluded that the tritium incor- porated into glycine is in a position analogous to the .-CHZOH group of L-serine. The data given in Table V on the cleavage of n-allothreonine lead to an analogous conclusion. However, the decarboxylation of aminomalonate in tritiated water yields glycine-2-t only about half of whose tritium can be released by n-amino acid oxidase. This indicates that the product is a mixture of R-glycine-2-t and S-glycine-2-t. This lack of stereospecificity is confirmed by the studies with specifically carboxyl- labeled [14C]aminomalonate (Table VI). The present data (Table IV) and earlier findings (24, 25) indicate that serine- hydroxymethylase can interact with several n-amino acids. It thus appears that aminomalonate can react with the enzyme both as a n-amino acid and as an L-amino acid (as indicated in Fig. 9) so that either one of the carboxyl groups may be removed in the reaction, which thus leads to the formation of both R- and S-glycine-2-t. This result is in striking contrast to that which obtains with L-aspartate /3-decarboxylase, which decarboxylates aminomalonate in tritiated water stereospecifically to yield S-glycine-2-t (2) ; it is notable that this enzyme exhibits strict n-amino acid specificity (37). Thus, the decarboxylation of aminomalonate to glycine occurs in a specific manner when catalyzed by one enzyme (aspartate P-decarboxylase) and in a nonspecific manner when catalyzed by another (cytoplasmic serine hydroxymethylase) .

10. 11. 12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22. 23.

24.

25.

26.

27.

28.

29. 30.

In early investigations on the serine-glycine interconversion, aminomalonic acid was considered as a possible intermediate (48- 50), but this idea was discarded on the basis of studies with labeled compounds and because aminomalonate is a symmetrical compound (51). The very considerable amount of information now available about the serine-glycine interconversion and the participation of tetrahydrofolate in this reaction (52), as well as the absence of evidence for the formation of aminomalonate from serine, render unlikely the possibility that aminomalonate is an intermediate between serine and glycine. The present studies, which indicate that the decarboxylation of aminomalonate in liver does not occur in a specific manner, would seem, in retrospect, to support Shemin’s (51) original conclusion (inadequate though it was on logical grounds, as pointed out by Ogston (1)) that aminomalonate could not be an intermediate in the conversion of serine to glycine.

31. 32. 33.

34.

35.

36.

37. TATE, S. S., AND MEISTER, A. (1971) Advan. Enzymol. 36, 503- 543

38. NEWTON, W. A., MORINO, Y., AND SNELL, E. E. (1965) J. Biol. Chem. 240, 1211-1218

39. BRAUNSTEIN, A. E., GORYACHENKOVA, E. V., TOLOSA, E. A., WILHARDT, I. H., AND YEFREMOVA, L. L. (1971) Biochim. Biophys. Acta 242, 247-260

40. RIARIO-SFORZA, G., PAGANI, R., AND MARINELLO, E. (1969), Eur. J. Biochem. 8, 88-92

41. MARTINEZ-CARRION, M., CRITZ, W., AND QUASHNOCI<, J. (1972) Biochemistry 11, 1613-1616

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Anil G. Palekar, Suresh S. Tate and Alton MeisterSERINE HYDROXYMETHYLASE AND ALLOTHREONINE ALDOLASE

Rat Liver Aminomalonate Decarboxylase: IDENTITY WITH CYTOPLASMIC

1973, 248:1158-1167.J. Biol. Chem.

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Rat Liver Aminomalonate Decarboxylase