d-alanine-d-glutamate transaminasethorne et al. (2,3) discovered t,hat n-amino acids could undergo...

THE JOURNAL OF R~O~OOICA~. CHEMIBTRY Vol. 240, No. 9, September 1966

Printed in U.S.A.

D-Alanine-D-Glutamate Transaminase

I. PURIFICATION AND CHARACTERIZATION*

M. MARTINEZ-CARRION~ AND W. TERRY JENKINS

From the Department of Biochemistry, University of California, Berkeley, California 947.20

(Received for publication, January 4, 1965)

Thorne et al. (2,3) discovered t,hat n-amino acids could undergo traneamination reactions with extracts of Bacillus subtilis and Bacillus anthracis. They proposed that. the biosynthesis of the cell wall n-glutamate, in fact, occurred solely via the transamination of n-alanine with ar-ketoglutarate. The participation of n-amino acids in transamination reactions was demonstrated subsequently in other microorganisms (3-5).

The existence of transaminases specific for the n-amino acids thus poses four major questions. (a) How many such enzymes are present in a particular organism? (b) How many of the n-amino acids synthesized by the organism are made solely by transamination from the corresponding keto acid (6)? (c) In what ways do the transaminases specific for the n-amino acids either resemble, or differ from, those for the L isomers? (d) What relationship do these enzymes bear to other pyridoxal phosphate enzymes?

The present papers, which are concerned with the extensive purification and characterization of n-alanine-n-glutamate transaminasel from B. subtilis, are an approach to answering some of these questions.

EXPERIMENTAL PROCEDURE

Materials and Methods

DEAE-Sephadex was purcha.sed through Pharmacia. Hy- droxylapatite was prepared by a modification of the method of Tiselius, HjertPn, and Levin (8). Lactic dehydrogenase was purchased from C. F. Boehringer.

Growth of Bacteria

Bacillus subtilis (NRRLB 1471)* was grown essentially by the procedure described by Thorne et al. (2, 3) in lo-liter batches which were incubated for 12 hours after inoculation with 300 ml of medium. This medium had been inoculated from a nutrient agar slant 12 hours previously. The cells were grown in a New Brunswick fermenter with aeration; foaming was prevented by the addition of 1 ml of General Elect#ric Antifoam 60 per liter of medium. The cells were harvested with a steam-driven Sharples

* Supported in part by Research Grants HO4417 from the United States Public Health Service and G20108 from the National Sci- ence Foundation. A preliminary note of some of this work has been published (1).

t Present address, Istituto di Chimica Biologica, University of Rome, Italy.

1 Also called D-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.10) (7).

* A gift of Dr. R. A. J. Warren.

supercentrifuge. Approximately 7 g of cells (wet weight) were obtained per liter of medium.

Enzyme Assays

The two assays used were both based on the determination of the pyruvate formed when n-alanine reacts with a-ketoglutarate.

Assay A-A calorimetric determination of pyruvate, based upon its reaction with salicylaldehyde in alkali (9), was con- sidered satisfactory for the measurement of activity throughout the enzyme purification procedure. The reaction was stopped by the transfer of 1 ml of the reaction mixture into 1 ml of 60% KOH. A 0.5-ml portion of 2% salicylaldehyde in absolute ethanol was then added, and the orange color was developed by a further incubat.ion at 37” for 10 min. Color development was stopped by the addition of 2.5 ml of cold water. The absorption at 480 rnp was found to be proportional to the enzyme concentration (Fig. 1). None of the other components of the assay mixture interfered with this calorimetric assay.

Assay B-This was a more sensitive spectrophotometric assay in which pyruvate product,ion was coupled to DPNH oxidation with lactic dehydrogenase. The decrease in absorbance at 340 rnp was proportional to the enzyme concentration (Fig. 1). This assay also gives a straight line in the absence of added pyridoxal phosphate.

Protein Determination

Protein concentrations were determined by the procedure of Lowry et al. (10). Crystalline bovine serum albumin was used as a standard.

Units of Enzymatic Activity

A unit of enzyme activity is defined as the amount of enzyme which produces 1 pmole of pyruvate per min in Assay .4. The specific activity of the enzyme is defined as units per protein equivalent to 1 mg of bovine serum albumin.

Sedimentation Constant

The enzyme used for this purpose had a specific activity of 90 and was concentrated to 8 mg per ml as follows. A solut,ion containing 2 mg of enzyme per ml, previously equilibrated with 0.02 M potassium phosphate buffer, pH 7.0, was placed in 8/100 Visking dialysis tubing. The tubing was suspended in a vacuum flask in an ice bath, and a steady vacuum was applied to the outside of the tubing.

The sedimentation velocity was determined with a Spinco model E ultracentrifuge equipped with a phase plate as a schlieren

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September 1965 M. Martinez-Carrion and W. T. Jenkins 3539

diaphragm and an automatic photoelectric scanning absorption system developed by Schachman et al. (11, 12). Double sector 12-mm cells equipped with quartz windows were used. The experiment was conducted at, 2.9” with a rotor speed of 59,780 rpm.

&‘olecular Weight

The apparatus and the enzyme solution used to determine the molecular weight of II-alanine-n-glutamate transaminase were identical with those employed for the measurement of the sedimentation constant,. The met,hod used was the sedimentation equilibrium pattern of the material absorbing at 404 rnp (13). The rotor was first accelerated to 20,000 rpm for 2 hours and t,hen maint,ained at 10,598 rpm for 16 hours at 2”.

Free Boundary Electrophoresis

h protein solution with a specific activity of 90 was used for the electrophoretic runs. The protein sample, 6.6 mg in 5 ml, was dialyzed for 8 hours against 4 liters of potassium phosphate buffer, pH 7.0, and then concentrated by the vacuum technique, described previously, to a final concentration of 3 mg per ml in a total volume of 2.2 ml. A Perkin-Elmer (model 38) free boundary electrophoresis apparatus was employed with a 2-ml cell. The temperature was 4” and the buffer 0.05 M potassium phosphate, pH 7.0. A current of 11.5 ma (130 volts) was applied.

Starch Gel Electroph,oresis

The apparatus described by Smithies (14) and the discontinu- ous buffer system of Barret, Friesen, and ,4stwood (15) were used. Direct current was supplied by a Heathkit regulated power supply.

A 50.~1 enzyme solution (0.5 mg of protein) was placed in one slot, and a 50-~1 bovine serum albumin solut.ion (0.2 mg of protein), as a standard reference, was placed in another. The gel was covered with Saran Wrap to prevent evaporation. Contact between the gel ends and the electrode vessels was made through four layers of Whatman No. 1 paper saturated with the electrode solution. The electrode vessels contained 1 liter each of a buffer made of 0.1 M lithium hydroxide and 0.38 M boric acid. A current of 30 ma (450 volts initially) was passed for 4 hours through the horizontal gel block in a 4” cold room.

After completion of the electrophoretic run the gel was sliced horizontally, and the top portion was stained with a 0.050/, solution of nigrosine in methanol-acetic acid-water (50 : 10 : 40) for 1 hour. After decolorizing overnight with 0.009% nigrosine in the same solvent, all protein bands were plainly visible against a clear background. The distance from the origin to the protein bands was measured and, making allowances for the shrink- age undergone in the stained half, equivalent band widths were cut out of the unstained half. These were triturated with 2-ml aliquots of 0.02 M potassium phosphate buffer, pH 7.0. After removal of the insoluble starch by centrifugation, the supernatant solutions were assayed for act’ivity by Assay A.

Substra,te Specificity

The ability of different I)- or L-amino acids to transaminate with oc-keto acids was measured in 0.5-ml volumes containing 50 Mmoles of Tris buffer (pH 8.3), 20 pmoles of the amino acid,

and 20 pmoles of the oc-keto acid. ,4fter the addition of 2 pg of enzyme the reaction was allowed t,o proceed for 30 min at 37”.

i o.2,-/o , j 5 4.5 13.5 180 5. n pg protein

,ug protein

FIG. 1. Relationship between micromoles of pyruvat)e formed and amount of enzyme. A, measurement of pyruvate formation by the salicylaldehyde assay. The 2-ml reaction mixture consisted of 50 mM Tris buffer (pH 8.3), 25 mM D-alanine, 10 mM a-ketoglutarate, 10 pg of pyridoxal phosphate per ml, and varying amounts of an enzyme preparation of a specific activity of 37 units per mg of protein. Incubation time was 10 min at 37”. B, measurement of pyruvate formation by the lact,ic dehydrogenase assay. The l-ml reaction mixture consisted of 100 mM Tris buffer (pH 8.3), 50 mM n-alanine, 20 mM a-ketoglutarate, 20 pg of pyridoxal phosphate per ml, 0.2 mM DPNH, an excess of lactic dehydrogenase, and enzyme with a specific act,ivity of 90 units per mg of protein at a temperature of 37”.

Enzymatic reactions were then st,opped by placing the tubes in a boiling water bath for 5 min. The amount of a new amino acid formed was det’ermined aft’cr paper chromatography by the quantitative ninhydrin procedure of Kay, Harris, and Enten- man (16).

Resolution of Transaminase

To resolve the enzyme from its cofactor, 2.5 mg of the enzyme in a 2-ml volume, 0.2 M with respect to potassium phosphate buffer, pH 5.2, containing 0.02 M u-alaninc were left at room

temperature for 15 min. Ammonium sulfat,e was added to give 40% saturation, and the precipitat,e that formed was collected by centrifugation. It was suspended in 0.2 ml of the same buffer, and the previous operation was repeated. The precipitate was suspended in 2 ml of 0.5 M potassium phosphate, pH 5.2, and dialyzed for 4 hours against 4 liters of 0.01 M potassium phosphate buffer, pH 7.0. The preparation then showed some cloudiness which was removed by centrifugation.

Reconstitution of Holoenzyme

To reconstitute t’he resolved enzyme, the apocnzyme was divided into two l-ml portions, and 10 1.19 of pyridosal phosphate were added to one portion while 10 pg of pyridosamine


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3540 D-Alanine-D-(-rutamate l’ransaminase. I Vol. 240, No. 9

TABLE I pro&mine sulfate adjusted to pH 7.0. After 15 min the solution Purijication of B. subtilis o-alanine-o-glutanlate transaminase was centrifuged for 45 min at 40,000 x g in a Spinco preparative

Fraction No. and:step Volume

I. Cell-free extract. II. Protamine sulfate..

III. First ammonium sulfate.

IV. First DEAE-Sepha- dex...............

V. Hydroxylapatite. VI. Second DEAE-Seph-

adex. . VII. Second ammonium

sulfate..

ml

2680 3500

425

940 275

5500

2700 1300

1200

1120

50

2.9 0.8

0.31

5.6

0.26

1.0 8.0

80

90

48 15 $8- ts

2.2 13 fi a, 0.6- - z x

Total units

-

7800 7000

I ultracentrifuge. The sunernatant fluid had an absorbance Protein

m/ml

37 20

Specific activity

units/?%?

0.078 0.10

Yield ratio (Aulo:iZSO) of 0.8 (Fraction II, Table I).

% Step III: First Ammonium Xulfate Fractionation-Finel:

100 ground ammonium sulfate, 263 g per liter of solution, was added

90 to 3.5 liters of Fraction II. After 15 min at 4”, the precipitate was removed by centrifugation. An additional 132 g of ammo-

70 nium sulfate per liter of the supernatant fluid were then added.

phosphate were added to the remaining l-ml portion. The solutions were immediately dialyzed in separate containers against 2 liters of 0.05 M potassium phosphate buffer, pH 7.0.

Reduction of Bound Pyridoxal Phosphate by Sodium Borohydride

A solution containing 1.3 mg of enzyme (specific activity, 90) per ml was treated with 0.005 M sodium borohydride for 5 min Volume of eluate(ml.)

by the dialysis method of Matsuo and Greenberg (17). The FIG. 2. Chromatography of Fraction V on DEAE-Sephadex. enzyme solution was then dialyzed for 9 hours against 1 liter of Conditions are given in the text.. l , absorbance at 280 rnp; and 9.04 M potassium phosphate buffer, pH 7.0. A, n-alanine-n-glutamate transminase activity, determined by

the salicylaldehyde assay. Determination of Transaminase Activity between Pyridoxamine

and Pyruvate or a-Ketoglutarate TABLE II

Enzyme having a specific activity of 40 in the absence of added Specijicity of amino acids as amino group donor

pyridoxal phosphate and of 90 in the presence of pyridoxal phosphate was used for this determination. Reaction mixtures containing 6 InM pyridoxamine, 10 mM keto acid, 50 mM EDTA, 200 mM Tris buffer (pH 8.2), and 0.22 mg of enzyme in a volume of 1 ml were incubated at 37” for 1 hour. Aliquots were then assayed for pyridoxal formation by the phenylhydrazine method of Wada and Snell (18).

RESULTS

Purijkation of Enzyme

All operations were carried out at O-5”. Enzyme preparations could be frozen and thawed at any stage of purity. The purification procedure and the results of a typical preparation are summarized in Table I.

Step I: Cell-free Extracts-Each batch of B. subtilis (about 70 g, wet weight) was suspended in 300 ml of 0.02 M potassium phosphate buffer, pH 7.0. The suspension was treated, 50 ml at a time, in a Raytheon sonic oscillator for 10 min and then centrifuged. The precipit,ate was discarded. The supernatant fluid, which had an absorbance ratio (AB0:A& of 0.58, is referred to as the cell-free extract. It was stored at -15” and thawed when needed (Fraction I, Table I).

Step II: Removal of Nucleic Acids wit?L Protamine Sulfate-To 2,680 ml3 of cell-free extract, obtained from about 1 Kg of B. subtilis (wet weight), were added 1,000 ml of a 1 y0 solution of

3 This preparation has been carried out on smaller and larger scales. The volumes of fractions, reagents, resin bed volume and columns, and gradient volumes were changed proportionally.

Amino acid donor

n-Alanine ............... n-Glutamate. ........... Da-Aminobutyrate ...... n-Aspartate ............. u-Asparagine ........... n-Norvaline. ............ n-Norleucine. ........... n-Ornithine ............. n-Methionine ........... n-Serine ................ n-Threonine, n-cysteine,

n-tryptophan, D-leu- tine, n-isoleucine, D- lysine, n-valine, n-phe- nylalanine ............

Glycine ................. L-Alanine, L-serine, L-

threonine, L-valine, L- aminobutyrate, L-aspartate ...............

L-Glutamate. ........... DL-Aminopimelic acid,

DL-aminoadipic acid. DL-cu-Methylserine. ......

i

Keto&!;tarate

pdes D- glutamate

3.8

3.8 1.56 2.35 1.9

Trace 0.25 0.52 0.26

0* 0

0 0

0 0

Keto acid acceptor

Pyruvate

- I Ketob:;yrate

pm&s D- alanine

2.5 6.4 2.25 1.3 1.72

Trace 0.25 0.73

Trace

i

,,moles D-u- aminobutyrale

5.3 2.1

* Indicates no detectable activity under these assay conditions.


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September 1965 M. Martinez-Carrion and TV. T. Jenkins 3541

0.83 A = Schlieren 0 =404

0.81 x

3

079

I I I 40 80 120

minutes

FIG. 3. Sedimentation velocity pattern of n-alanine-n-glutamate transaminase at an initial concentration of 8 mg per ml in 0.02 M potassium phosphate, pH 7.0. Top, schlieren pattern taken 108 min after attaining a speed of 59,780 rpm at a temperature of 2.9”. Sedimentation is proceeding from the meniscus on the left toward the right. Center, absorbance changes across the cell measured concurrently at 404 mp. The sigmoid shaped curve in the center represents the sedimenting chromoprotein. The spikes to

After 15 min the precipitate which formed was collected by centrifugation and dissolved in about 200 ml of potassium phosphate buffer, 0.03 M, pH 7.4 (Fraction III, Table I).

Step IV: First DEAE-Sephadex Chromatography-A column (4 X 44 cm) was prepared from 40 g of DEAE-Sephadex (A-50) equilibrated with 0.03 M potassium phosphate buffer, pH 7.4. Fraction III was dialyzed overnight against 12 liters of 0.03 M potassium phosphate buffer, pH 7.4, and then applied to the DEAE-Sephadex column at a rate of 250 ml per hour. The adsorbent was washed with 1000 ml of the same buffer. This washing contains much protein but only about 12 to 15% of the enzymatic activity. The adsorbed protein was then eluted at a flow rate of 66 ml per hour with a linear gradient made from 1.5 liters of 0.03 M potassium phosphate, pH 7.4, in the mixing bottle, and 1.5 liters of 0.4 M NaCl and 0.2 M potassium phosphate buffer, pH 7.1, in an identical reservoir bottle. Fractions of 22 ml were collected. Approximately 60%, and sometimes as much as 75%, of the applied enzyme was obtained in the eluate. The fractions containing enzyme with a specific activity ranging from 0.4 to 5.2 units per mg of protein were pooled (Fraction IV, Table I).

Step V: Hydroxylapatite Chromatography-To Fraction IV, 480 g of ammonium sulfate per liter were added with stirring, and the precipitate was removed by centrifugat,ion. This precipitate u-as dissolved in 30 ml of 0.005 M potassium phosphate buffer, pH 7.0, and dialyzed overnight against 8 liters of the same buffer. The dialyzed material was applied to a calcium hydroxylapatite column (4 X 35 cm) previously equilibrated with 0.005 M potassium phosphate buffer, pH 7.0. The adsorbent was washed with 300 ml of the same buffer. The concentration of potassium phosphate buffer, pH 7.0, was then increased linearly to a limit of 0.07 M after 2 liters. The flow rate was approximately 80 ml per hour; fractions of 20 ml were collected; and 60% to 85% of the applied enzyme units were eluted. The fractions containing enzyme with a specific activity ranging from 4 to 11 units per mg of protein were pooled (Fraction V, Table I).

Step VI: Second DEAE-Sephadex Chromatography-Ammo- nium sulfate (480 g per liter) was added to Fraction V. The precipitate was removed by centrifugation. This precipitate was dissolved in 10 ml of 0.04 M potassium phosphate buffer, pH 7.1, and dialyzed overnight against 8 liters of the same buffer. A column (24 X 2 cm) of DEAE-Sephadex (A-50) was prepared and equilibrated with a buffer identical with the one used for dialysis. Dialyzed Fraction V was applied to the DEAE- Sephadex column and immediately washed with 0.08 M potassium phosphate buffer, pH 7.1. Elution was then started with a gradient formed with 500 ml of 0.08 M potassium phosphate buffer, pH 7.1, in the mixing chamber and 500 ml of 0.3 M KC1 and 0.15 M. potassium phosphate, pH 7.1, in an identical reservoir. Both reservoir and mixing bottles were at atmospheric pressure. The flow rate was 26 ml per hour and 6-ml fractions were collected (Fig. 2). Of the applied activity units 99% were ac- counted for. The fractions containing enzyme with a specific activity ranging from 30 to 95 units per mg of protein were pooled (Fraction VI, Table I).

Step VII: Second Ammonium Sulfate Fraction&on-The 48

the left of this on the base-line are due to the meniscus. Bottom, the distances of the boundaries shown in A and B from the center of rotation (X) are indicated as a function of time.


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3542 D-Alanine-~-Glutamate Transaminase. I Vol. 240, No. 9

rs,

FIG. 4. Sedimentation equilibrium pattern of n-alanine-n-glutamate transaminase. The solution originally contained 8 mg of enzyme per ml in 0.02 M potassium phosphate buffer.

FIG. 5. A, free boundary electrophoresis pattern. Photograph of the descending electrophoretic boundary of a 0.3% solution of the purified transaminase. The picture was taken 30 min after initiation of the experiment. The “peak” on the left is at the position of the original boundary and is due to the buffer. B, starch gel electrophoretic patterns of 0.2 mg of bovine serum albumin (upper) and 0.5 mg of n-alanine-n-glutamate transaminase preparation (lower). Migration is toward the anode on the right.

ml of Fraction VI were reduced to 24 ml with an LKB 6300 A ultrafilter, and 15 g of powdered ammonium sulfate were added to this concentrated solution. The precipitate was removed by centrifugat,ion. To the supernatant fluid an additional 4.8 g of powdered ammonium sulfate were added. The precipitate, after cent.rifugation, was dissolved in 2 ml of 0.1 M potassium phosphate buffer, pH 7.0 (Fraction VII, Table II). This purified enzyme was stored at -10’.

The enzyme was stable at 4” for 1 month in 30% ammonium sulfate. It was stored at -10’ for 2 months in 0.02 M pota.s- sium phosphate buffer, pH 7.0, without loss of activity in spite of repeated freezing and thawing.

Physkochemicu.1 Properties of Enzyme

When the purified enzyme was examined in the analytical ultracentrifuge it was found, as is shown in Fig. 3, that the rates of sedimentation of the material absorbing at 404 rnp and of the principal peak in the schlieren pattern were identical. The sedimentation coefficient calculated for water at 20” and zero protein concentration was 4.278. The partial specific volume was assumed to be 0.74.

From the sedimentation equilibrium pattern (Fig. 4) a value of 53,000 f 4,000 for the molecular weight was obtained (12,13).

The same preparation of enzyme, when examined by free boundary electrophoresis presents a major component and traces of impurities (Fig. 5, upper). Starch gel electrophoresis indicated the same degree of homogeneity with the enzymatic activity in the region of the darkest band (Fig. 5, lower).

Absorption Spectra

Spectra of the purified enzyme, when recorded in a series of buffers in the pH range from 5.2 to 9.2, were found to be identical

o.oa-

0.07 -

0.06-

: f 0.05- d I3

0.04-

0.03-

0.02-

0.01 -

I 1 I I I 300 350 400 450 500

mtL

FIG. 6. Absorption spectra of the purified n-alanine-n-glutamate transaminase (0.1 mg per ml) in 0.1 M sodium tetraborate pH 9.2, and 0.1 M potassium phosphate, pH 5.2.


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within the experimental limits. Apart from the protein maxi- Vitamin Bg Content and Effect of Vitamin B6 Derivatives mum (280 mp), there are maxima at 330 rnp and 415 rnp (Fig. 6). Enzyme with a specific activity of 90 was subjected to the

Substrate Speci$city acid hydrolysis procedure of Rabinowitz and Snell (19) and was

The ability of this transaminase to catalyze amino group assayed microbiologically for vitamin Bs (20) by Dr. B. M.

transfer from different amino acids to cr-ketoglutarate, pyruvate, Guirard. An equivalent of 1 mole of vitamin Bg was found

and cr-ketobutyrate is presented in Table II. per 140,000 g of protein. Since this particular enzyme preparation was activated a-fold by the addition of pyridoxal phosphate,

Effects of Buffer and pH we can assume that it contained less than 50% active holoen- The enzyme, when examined in the presence of Tris, bar- zyme. .’

biturate, or phosphate buffers, has an optimum reactivity in the Transaminases from pig heart, such as n-glutamate+aspartate pH range of 8.2 to 8.8 (Fig. 7). No marked differences between (21) and n-alanine-n-glutamate (22), have been highly purified buffers were noted. and found not to exhibit an increase in maximum activity when

Equilibrium Studies incubated with pyridoxal phosphate or pyridoxamine phosphate. On the other hand, the glutamate-aspartate apoenzyme has

The approach to equilibrium of the reaction from both direc- been prepared, and it can be reactivated by the addition of tions was measured (Fig. 8). From these results the equilibrium either pyridoxal phosphate or pyridoxamine phosphate but not constant was calculated to.be 1.7.

0.05 .c 5 0.04

s fi k 0.03

k

a 8 2 0.02

4

0.01 -

07w 7 PH

by pyridoxal or pyridoxamine-(22, 23). Other transaminases,

3

I I I 00 120 160 minutes

FIG. 7 (left). Variation of enzymatic activity with pH. The Samples of 0.1 ml each were removed periodically, and the reao- spectrophotometric assay used is described in the text. 0, Tris, tion was stopped by addition of 100 pmoles of HClOd. Of this, 0.05 M; A, barbiturate, 0.1 M; 0, potassium phosphate, 0.1 M. 10-J aliquots were taken for pyruvate assay by the DPNH-lactic

FIG. 8 (right). Equilibrium of the reaction. The reaction in dehydrogenase method. For measurements of the reverse reac- the forward direction (0) was at 37” in a 3-ml volume in the pres- tion (O), the same method was used with the exception that 50 ence of 200 fimoles of Tris buffer (pH 8.3), 50 pmoles of D-glu- pmoles of a-ketoglutarate replaced pyruvate, and 50 pmoles of tamate, 50 pmoles of pyruvate, and 4 units of the purified enzyme. n-alanine replaced n-glutamate.

I I I 0 0.000 0.E 0.264

9 mM 10 minutes

FIG. 9 (left). Effect of varying concentration of pyridoxal phos- buffer (pH 8.3), 2 mu n-alanine, 10 mu cu-ketoglutarate, 0.16 mu phate, pyridoxamine phosphate, and pyridoxamine on n-alanine- DPNH, and an excess of lactic dehydrogenase at 37”. The reac- n-glutamate transaminase. To test the coenzyme specificity in tion was started by the addition of 0.5 pg of enzyme incubated at the purified n-alanine transaminase, the lactic dehydrogenase several time intervals with 20 pg of pyridoxal phosphate per ml assay was employed with different cofactor concentrations. The at 21” (-). Th e enzyme was also incubated for 9 min with 40 reaction was started by the addition of 0.5 rg of enzyme. mu of n-alanine before the actual incubation with pyridoxal phos-

FIG. 10 (right). Effect of incubating the enzyme with an excess phate (- - -). of pyridoxal phosphate. The mixture contained 100 rind Tris


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such as the tyrosine transaminase (24), after purification are stimulated by the addition of pyridoxal phosphate and pyridoxamine phosphate. ils shown in Fig. 9, the n-glutamate-n-aspartate transaminase of B. subtilis is also partially activated by added pyridoxal phosphate or pyridoxamine phosphate.

Pyridoxal phosphate and pyridoxamine phosphate were both good activators of the enzyme, but pyridoxal phosphate was better at low concentrations. At 0.088 mM, both were equally effective. Pyridoxamine did not produce any changes in the activity. Some preparations of purified enzyme exhibited 50% maximal activity in the absence of pyridoxal phosphate while in others it was as low as 25%.

The reactivation of glutamic-asparatate apotransaminase with pyridoxal phosphate takes about 10 min to achieve full recombi- nation (23). The effects of incubating the enzyme with an excess of pyridoxal phosphate were therefore studied (Fig. 10).

The enzyme was fully activated after 1 min of incubation, but, then a decrease in activity occurred. This was also true when the enzyme was incubated with pyridoxal phosphate in the presence of an excess of n-alanine.

Reduction of Enzyme-bound Pyridoxal Phosphate by Sodium Borohydride

Addition of sodium borohydride caused an immediate change in the spectrum. The 415 rnp peak disappeared, and there was a concomitant increase of the absorption at 330 rnp (Fig. 11).

This peak at 330 rnp did not change upon dialysis for 10 hours against 0.04 M potassium phosphate buffer, pH 7.0.

Similar spectral shifts had been observed with several pyridoxal phosphate-containing enzymes after treatment with sodium borohydride. Enzyme reduced with sodium borohydride re- tained only a trace of catalytic activity when assayed in the absence of added pyridoxal phosphate. Only half of the maximum activity appeared when pyridoxal phosphate was added to the assay mixture (Table III). The above data are inter- preted to show that the borohydride specifically inactivated

5-

3-

5-

)‘

I I I -

wavelength (mp)

FIG. 11. Absorption spectrum of D-alanine transaminase before and after reduction with sodium borohydride. Curve 1, spectrum of 1.3 mg of enzyme in 1 ml of 0.05 M potassium phosphate buffer, pH 7.0; Curve 9, spectrum of the same enzyme after reduction with sodium borohydride.

TABLE III Activity of enzyme before and after reduction with sodium

borohydride Assayed by Assay B; 10 rg of pyridoxal phosphate were present

when indicated.

Specific activity Enzyme preparation

+ Pyridoxal phosphate / - Pyridoxal phosphate

Before reduction. ........ After reduction ...........

92 44.5 40 1.2

FIG. 12. Spectral changes due to the pyridoxal phosphate moiety in the purified, resolved, and reconstituted enzyme. Curve 1, spectrum of the purified enzyme; Curve d, spectrum of the enzyme after addition of 40 pmoles of n-alanine; Curve 8, spectrum of the resolved enzyme; Curve 4, spectrum of the resolved enzyme after incubation with pyridoxal phosphate and dialysis against 2 liters 0.05 M potassium phosphate buffer, pH 7.0.

the holoenzyme, for it apparently did not affect the conversion of the apoenzyme, contained in the preparation, into active holoenzyme. We believe that, as shown for other pyridoxal phosphate proteins, it reduced only the azomethine linkage between the formyl group of the pyridoxal phosphate and an amino group on the protein.

Catalysis of Transamination of Pyridoxamk by Puti$ed Enzyme

The preparation of n-alanine-D-glutamate transaminase, which contained approximately 50% apoenzyme, used substrate keto acids as amino acceptors for transamination with pyridoxamine, as was shown for the glutamate-oxaloacetate apotransaminase from pig heart (25). After 1 hour of incubation 220 pg of enzyme produced 0.112 pmole of pyridoxal when a-keto- glutsrate was the amino acceptor and 0.072 umole of pyridoxal


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TABLE IV Enzymatic activity before and after resolution

I Specific activity*

pmozes pyruvate/min/mg protein % Before resolution. 16 52 72 After resolution. . 0.9 58 99.8 After reconstitution with pyr-

idoxal phosphate. . . 59 59 0 After reconstitution with pyr-

idoxamine phosphate. 28 56 56 51

* At a temperature of 23”.

when pyruvate was the acceptor. No detectable amount of pyridoxal was formed when pyridoxamine alone was incubated with the enzyme.

Resolution and Reconstitution of Enzyme

The absorption spectra of the enzyme in its purified, resolved, and reconstituted forms were measured. Upon the addition of n-alanine, the absorbance at 415 mp decreased and that at 330 mu increased. Both absorption maxima then disappeared when the enzyme was resolved but reappeared after reconstitution and removal of the excess B6 by dialysis. The ratio of absorbances at 280 rnp and 415 rnp in the reconstituted enzyme was 9 (Fig. 12).

Enzymatic activity and protein determinations were followed after each step of the resolution and reconstitution procedure (Table IV). The enzyme was completely resolved, but to reconstitute it only pyridoxal phosphate was completely effective under the conditions used.

DISCUSSION

The purified enzyme contains but a single yellow chromoprotein as indicated by both sedimentation velocity and sedimentation equilibrium ultracentrifugal analysis with monochromatic light (404 mp). These analyses also identified the chromoprotein as the major component in the preparation. Electrophoresis confirmed that this component accounts for more than 90% of the protein.

Treatment of the preparation with n-alanine and monobasic potassium phosphate caused the disappearance of both chromo- phore maxima and yielded inactive protein. Treatment with n-alanine alone yields a colorless protein with a maximum at 330 rnb which retains enzymatic activity. The addition of pyridoxal phosphate after the resolution restored both the catalytic activity and the characteristic spectrum. Microbio- logical determination of vitamin Bs indicated that 1 molecule was bound per molecule of protein when allowances were made for the amount of resolution of the enzyme during the purification procedure and the estimated purity.

Both 415 rnp and 330 rnp absorption maxima are therefore ascribed to the enzyme. Spectral maxima at 415 and 330 rnp have been associated with other pyridoxal phosphate proteins. They are believed to indicate that the pyridoxal phosphate is linked through its aldehyde group with an amino group on the protein. This hypothesis is supported by the characteristic

behavior upon reduction with borohydride. The most notable difference from the pig heart glutamic-aspartic and glutamic- alanine transaminases is the fact that the spectrum of this enzyme does not appear to change with pH. It differs in particular from the L-alanine transaminase of pig heart, which is a pH indicator (pK = 7.3) possessing an acidic form (hmax 425 mN) and a basic form which has a maximum absorbance at 330 mu with pronounced inflection points at 360, 390, and 420 mp.

It is of particular interest, however, that the L-leucine-glutamate transaminase of pig heart has a very similar absorbance curve with ma.xima at the same wave lengths and also does not act as a pH indicator in the range pH 5 to lo.* Preliminary evidence indicates that the only substantial spectral difference, in fact, is the sign of the optical rotatory dispersion Cotton effect.

The fact that serine transhydroxymethylase of rabbit liver is not a pH indicator is also of interest because it has a similar maximum at 430 rnM and has been shown to react very slowly, but specifically, with n-alanine to yield pyridoxamine phosphate and pyruvate (26). This reaction was shown to proceed through two intermediates with maxima at 430 rnp and 505 rnp, the latter being a basic species of the former (27).

From the results of Table II, it is apparent that this enzyme has a relatively narrow specificity. Only n-amino acids act as substrates, so that the preparation is free of any racemase. Moreover, under these conditions, the transaminase itself can- not act as a racemase.

This enzyme is fairly specific for n-amino acids with struct.ures similar to either n-alanine or n-glutamate. The participation of dicarboxylic amino acids in the reaction is not, however, es- sent,ial since a-aminobutyrate and alanine reacted with pyruvate. For transamination to proceed the substrate had to have a carbon chain more than 2 but less than 7. Thus glycine is inactive (with pyruvate), and there is a progressive decrease in reactivity ascending the homologous series n-a-aminobut,yrate, n-norvaline, n-norleucine. A linear carbon chain is favored since n-valine is inactive. The low activity with n-serine rela- tive to n-alanine also may be due to the branched configuration of the hydroxyl group.

SUMMARY

1. n-Alanine-n-glutamate transaminase was purified 1,150- fold from sonic extracts of Bacillus subtilis by standard protein fractionat.ion techniques. The preparations were about 95% homogeneous as estimated by the criteria of ultracentrifugation, free boundary electrophoresis, and starch gel electrophoresis. A value of 53,000 += 4,000 was obtained for the molecular weight by a sedimentation equilibrium method.

2. The purified preparation is yellow. Absorption maxima at 415 rnp and 330 rnp were ascribed to pyridoxal phosphate bound as prosthetic group to the n-transaminase. A characteristic single maximum is obtained at 330 rnl.c after reduction with sodium borohydride. The holoenzyme was estimated to contain 1 molecule of vit#amin Bs per molecule protein.

3. The preparative procedure described yields enzyme which is from one-third to two-thirds resolved from its prosthetic group, but it can be reactivated by the addition of pyridoxal phosphate or pyridoxamine phosphate.

4. The prosthetic group can be removed to form inactive apoenzyme from which holoenzyme may be regenerated by addi-

4 R. T. Taylor and W. T. Jenkins, unpublished observations.


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tion of pyridoxal and pyridoxamine phosphates but, not their unphosphorylated derivatives.

5. This enzyme reacts with only few substrates. n-Alanine, n-glutamate, n-aspartate, n-asparagine, and n-cY-aminobutyric and to a lesser extent n-methionine, n-serine, and n-ornithine are active. The L isomers are completely inactive. This preparation is also capable of transaminating pyridoxamine and pyruvate or cr-ketoglutarate but at a rate 1: 1000 of that of the transamination between n-alanine and a-ketoglutarate.

6. The transamination reaction between n-alanine and a-ketoglutarate has a pH opt,imum from 8.3 to 8.8 and a final equilibrium constant at pH 8.3 of 1.7 in favor of n-alanine formation.

Acknowledgment-We wish to thank Drs. H. K. Schachman and F. Putney for performing the ultracentrifugal analysis, Dr. B. M. Guirard for the microbiological assays for vitamin Bs, and Yiu-Hin Hui for assistance in preparing the enzyme.

REFERENCES

1. MARTINEZ-CARRION, M., AND JENKINS, W. T., Biochem. and Biophys Research Communs., 12,365 (1963).

2. THORNE, C. B., GOMEZ, C. G., AND HOUSEWRIGHT, R. D., J. Bacterial., 69, 357 (1955).

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6. KURAMITSU, H. K., AND SNOKE, J. E., Biochim. et Biophys. Acta, 62, 114 (1962).

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M. Martinez-Carrion and W. Terry JenkinsCHARACTERIZATION

d-Alanine-d-Glutamate Transaminase : I. PURIFICATION AND

1965, 240:3538-3546.J. Biol. Chem.

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