THE JOURNAL OP B~LCXXCAL CHEMISTRY Vol. 250, No. 11, Issue of June 10, pp. 4029-4036, 1975

Printed in U.S.A.

Purification and Properties of 7,8=Diaminopelargonic Acid

Aminotransferase

AN ENZYME IN THE BIOTIN BIOSYNTHETIC PATHWAY*

(Received for publication, August 21, 1974)

GERALD L. STONERS AND MAX A. EISENBERG

From the Department of Biochemistry, Columbia University, College of Physicians & Surgeons, New York, New York 10032

SUMMARY

The enzyme 7,8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precursor 7.keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7,8-diaminopelargonic acid. The enzyme has been purified nearly 1000.fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homo- geneous by the scanning of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values.

The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incuba- tion with the amino donor, S-adenosyl-L-methionine.

A molecular weight estimation of 94,000 f 10,000 has been obtained by gel tiltration and sucrose gradient sedimen- tation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47,000 f 3,000 indicating a dimeric enzyme.

A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equiva- lent to the 7,Sdiaminopelargonic acid produced in the reac- tion mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5’.methylthioadenosine and the neutral compound, 2.oxo-3.butenoic acid.

* This investigation was supported by Public Health Service Grant AM-14450 from the National Institute of Arthritis and Metabolic Diseases.

$ Part of this work represents a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree, Columbia University, predoctoral trainee under Public Health Service Trainine Grant GMO0255. Present address, Denartment of Microbiology aid Immunology, Albert Einstein Colle-ge of Medi- cine Bronx, New York 10461.

An early intermediate in the synthesis of biotin in Escherichia coli is 7-keto-8-aminopelargonic acid, a condensation product of pimeloyl-CoA and L-alanine (1). We have previously shown that 7-keto-8-aminopelargonic acid is transaminated in the pres- ence of S-adenosyl-L-methionine and a cell-free E. coli estract to 7,8-diaminopelargonic acid (2). Carbonylation of IIAPA’ to form the ureido ring and the subsequent addition of a sulfur atom to form the tctrahydrothiophene ring complete the syn- thesis of the bicyclic molecule (3) as shown in the following reac- tion sequence;

7.keto- PLP

pimeloyl-CoA + L-Ala F S-amino- Ado-Met, PLP

09 pelargonic (A) acid

7, %diamino pelargonic acid Ho-, ATP Dethiobiotin

CD) (B) ‘$”

Biotin

The letters under the arrows indicate the cistrons of t.he biotin operon of E. coli which specify the respective enzymes (4). The purification and characterization of 7-keto-8-aminopclargonic acid synthetase (the biol? gene product) (5) and dcthiobiotin synthetase (the bid> gene product) have been reported (6, 7). We now report the purification and characterization of the bioA gene product, to which we have given the trivial name DAPA aminotransferase.2 While this work was in the process of comple- tion, Itzumi et al. (8) reported on the purification of the DAPA aminotransferase enzyme from Brev-ibacterium divaricatum. Al- though the enzyme was purified 5000.fold, it had only SOY, of the specific activity of our enzyme. Neither the properties of the enzyme nor the kinetics of the reaction were discussed

Since this is, to our knowledge, the only transaminase reaction which utilizes Ado-AVet as an amino donor, extensive purification of DAPA aminotransferase was undertaken in order to establish the following: (a) that the observed activity is, in fact, due to a single enzyme and therefore Ado-Met interacts directly with

1 The abbreviations used are: DAPA, 7,8-diaminopelargonic acid; Ado-Met, S-adenosyl-L-methionine; PLP, pyridoxal phos- phate; PMP, pyridoxamine phosphate.

* The systematic name for this enzyme would be S-adenosyl- L-methionine:7-keto-%aminopelargonic acid aminotransferase.

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DAPA aminotransferase, and (b) that the previously observed

ability of ATP and L-methionine to replace Ado-Met in the cclI-

free system (2) was due to the intermediate formation of Ado-

Met and not to an intrinsic activity of DAPA aminotransferase

with these substrates. In addition, experiments with the purified

enzyme and radioactively labeled Ado-Met have permitted the

detection and quantitation of a degradation product of Ado-Met

deamination indicating the lability of the keto product formed

from Ado-Met.

EXPERIMENTAL PROCEDURE

Materials

7-Keto-%aminopelargonic acid and 7,8-diaminopelargonic acid were synthesized by the methods previously reported (4). L- Methionine, S-methyl-L-methionine, tris(hydroxymetjhyl)amino- methane (enzyme and buffer grade), ammonium sulfate (enzyme and buffer grade), sucrose (enzyme grade), ovalbumin, and y- globulin (human) were obtained from Schwarz/Mann. The fol- lowing were obt,ained from Sigma: S-adenosyl-L-ethionine, S- adenosyl-L-homocysteine, ATP, PLP, PMP, dithiothreitol, aldol- ase, yeast alcohol dehydrogenase, and protamine sulfat,e (grade I). Malate dehydrogenase, fumarase, and L-amino acid oxidase (Crotalus terrificus terri$cus) were obtained from Boehringer Mannheim. Alkaline phosphatase (Escherichia coli) was a product of Worthington. Bovine serum albumin was from Pentex. Thiamine hydrochloride and 2.mercaptoethanol were supplied by Eastman.

All of the chemicals required for the preparation of polyacryl- amide gels, including sodium dodecyl sulfate, were obtained from Canalco. DEAE-cellulose (DE52) and cellulose phosphate (Pll) were products of Whatman. Sephadex G-100 and (i-200 were sup- plied by Pharmacia. Bio-Iiad was the source of hydroxylapatite and Dowex 50 (AC 5OW-X8, 200 to 400 mesh).

DL-[2-%]Methionine was supplied by New England Nuclear. All of the other chemicals were reagent grade.

Media-Tryptone broth was prepared as follows: 10 g of Bacto- tryptone (Difco) and 2.5 g of NaCl were autoclaved in 1 lit,er of glass-distilled water and 0.1 ml of a 0.570 (w/v) solution of thia- mine hvdrochloride was added asenticallv. The trvptone-maltose

_I

medium utilized for growth of 50.iiter batrhes of’iiacteria in the fermenter contained in 48 liters of deionized water: 500 g of Bact)o- tryptone, 250 g of NaCl, 125 g of MgS04.7Hz0, and 25 ml of 10% NaOH. After sterilization in the fermenter, 500 ml of filter-stcri- lized 20% maltose solution was added asept,ically.

Bacterial Strains-A regulatory mutant, which is not repressible by biot,in, referred to as strain 10, was utilized for the purification of DAPA aminotransferase. This strain was selected from parent, strain E. coli K-12 strain YlO-1 by virtue of its resistance to the antibiotic action of the biotin antagonist a-dehydrobiotin (3).

Methods

Growth of Cells-Cells of E. coli strain 10 were grown overnight in 2 liters of tryptone broth. These cells were the innoculum for 50 liters of the tryptone-maltose medium contained in a loo-liter capacity fermenter (model F-130, New Brunswick Scientific Co.). The cells were grown with stirring (200 rpm) and aeration (5 liters/min) to lace log phase (200 Klett units) and then harvested bv centrifunation (CEPA. Karl Padberg. Germ:Lny). The yield of

d . , .

cells was from 5 to 7 g (wet weight) per liter. DAPA Aminotransjerase Assay-The standard assay condit,ions

have been described (2). One unit of enzyme activity is now de- fined as that amount which produces 1 pmol of l>APA per min under the standard conditions. The specific activity is the number of units per mg of protein.

A simplified assay which was employed to locate the enzyme activity in column chromatography fractions was carried out as follows: 10 or 20 ~1 ot the fraction was added to 100 ~1 of a sub- strate mixture consisting of 0.15 M Tris-Cl (pH 8.5), 5 mM dit,hio- threitol, 0.2 rnM PLP, 0.01 rnM 7-keto-8.aminopelargonic acid, and 2.5 mr-,z Ado-Met. After a 15.min incubation at 37”, 50 ~1 of 150; trichloracetic acid was added to stop the reaction, and a 10-J sample was removed for bioassay.

Btoassay of DAPA-DAPA was quantitatcd by the disc assay procedure previously described (2). The assay organism, E. coli strain bioA109 is a biotin auxotroph which is defective in DAPA

aminotransferase and t,hus can utilize DAPA, but not 7-keto-8. aminopelargonic acid as a growth factor.

Protein Dele?rr2ination-Determination of protein in the early stages of the l>APA aminotzransferase pllrification was arrording to the method of Lowry et al. (!,) with bovine serllrn albumin as the standard. In the final t,wo stages of the enzyme purification, the low protein concentration (less than 100,~g;ml) and the inter- ference by 2-mercaptoethanol and glycerol which were present in the enzyme solution precluded the use of the Lowry procedure. Therefore, a modificatjion of the light scattering method of Tap- pan (IO) was used. The protein was precipitated by the addition of 1 volume of the reagent (24yc acetic acid and 4.27, K3Fc(CN)6) to 5 volumes of the enzyme solution. The scattered light was de- termined 5 min aft.er the addition of the reagent. in a microcell in the Perkin-Elmer Fluorescence Spectrophotometer (hlodel MPF- 2A) with both the excitation and emission wavelengths set at 480 nm. Bovine serum albumin prepared in the enz)me buffer was used as a standard. With this method, protein concentrations as low as 10 pg/ml could be measured.

Determination of Radioaclivity-liadioactivity was determined with an Ansitron or a Beckman model LS-230 liquid scintillation counter. The scintillation fluid has been described (6). Aquas01 (New England Nuclear) was used as the scintillation fluid for liquid samples.

Preparation and Anally&s of S-Adenosyl-L-I~lcthionine-Ado- Met of higher purity than available commercially was prepared by the perchloric acid extract,ion of yeast according to the method of Schlenk, et al. (11). The Ado-Met was purified on a column of Dowex 50-H+ by a modification of the method of Schlenk an,d Ehninger (12). The purity of Ado-h,Iet was established by thin layer chromatography with 1-butanol-acetic acid-water (GO: 15:25) as developing solvent. On the basis of absorption at, 260 nm (13) the preparation was 05 to 98’y0 pure.

Pkpnralion of S-Adcnos~~l -L- (2-hytlrory-4-7lleth!/lthio)butyric Aci&-The 2.hvdroxv derivative of Ado-Met was prepared by ” ” nitrous acid deamination according to the method hf %appia et al. (14). A small amount of the doubly-deaminated derivative of Ado-Met. S-inosvl-L-(2.hvdroxv-4.methvlthio)butvric acid was I ," I _ ." obtained as a by-product, of this procedure.

Preparation of S - A&TLOS!J~ - I. [2 -IT] rncthionine-Ado -Met la- beled in the 2.position of the met,hionine moiety was prepared enzymatically from 200 PCi (7.6 mg) ot,-/2-‘“C]methionirle. Mcthio- nine adenosyl transferase from E. coli B was prepared according to the procedure of Takor and Tabor (15). The reaction mixture employed was that described by Lombardini et al. (IG), except that 0.013 M NaCN was included to inhibit the derarboxylation of the newly synthesized Ado-Met. The S-adenosyl-L-[2-14C]methio- nine was purified as previously described. The product on anal- ysis by thin layer chromatography showed only one ultraviolet- absorbing compound, which also contained !)8.5% of the radioac- tivity. Unlabeled Ado-Met was added to give a specific act.ivity of 2.32 X lo6 cpm/fimol.

Polyacrylamide Disc Gel Electrophoresis-The purification of DAPA aminotransferase was monitored by polyacrylamide disc gel elcctrophoresis in 79& gels (pH 9.5) prepared according to the method of Davis (17). Nonstandard gels, a 15y6 gel rlmning at pII 9.5 and a 7% gel running at pl1 8.0, were prepared according to Gabriel (18).

Molecular Weight Estimation-The molecular weight of the aminotransferase was determined by three procedures; ((0 poly- acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to the method of Weber and &born (lo), (b) gel filtration on Sephadex G200 by a modification of !.he method of Andrews (20), (c) sucrose gradient, centrifugation by a modifica- tion of the method of Martin and Ames (21).

Isoelectric Focusing in S?lcrose Density Gradient-An LKB iso- electric focusing apparatus (110-ml capacity) was first utilized with l(rO wide range (pH 3 to 10) ampholytes. In a second experi- ment, the ampholyte- coIlcerltrittion.wits- increased to 57,, while the nH ranee was narrowed to ~11 3 to 6. In addition, O.l%, 2- <, mercaptoethanol was added to the gradient to stabilize the-en- zyme.

Purification

In the sequence of the purification steps described below all of the buffers were prepared with glass-distilled water. Determina-

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tions of buffer pH were made at ambient temperature. All pro- cedures were carried out at O-4”, except where noted.

Step 1: Preparation of Cell-jree Extract-The enzyme was pre- pared from 75-g cells of E. coli strain 10 which were grown as de- scribed under “Methods” and stored at -20” until used. The en- zyme activity was found to he stable at -20” in the cells or at any stage of the purification for periods of up to 1 year or more. How- ever, in the latter stages of the purification, the enzyme must he protected by freezing in the presence of 207, glycerol. The extract was prepared by sonic disruption (Branson, model W14OD) of a suspension of the unwashed cells in Buffer A (0.05 M potassium phosphate buffer (pH 7.0), containing 10 mM 2-mercaptoethanol, 1 mM PZDTA, and 0.05 mM PLP (4 ml of bilffer per g cells)). The sonic extract was centrifuged at 30,000 rpm for 1 hour (30 rotor, Beckman, model L).

Step 2: Protamine Sulfate Precipitation of Nucleic Acids-The protamine sulfate (0.25 ml of a 27’ solution prepared in Buffer A per ml of extract) was added dropwise while-the extract was con- tinuously stirred. The stirring was continued for 20 min after the addition- was complete. The-mixture was centrifuged at 12,000 rpm for 30 min (GSA rotor, Sorvall RC2-B).

Step 3: Ammonium Sulfate Fractionation-The protein fraction precipitating between 35 and 557, ammonium sulfate sat,uration was prepared as follows. To the protamine-treated supernatant (300 ml) was slowly added powdered ammonium sulfat,e (62.7 g) while the mixture was continllonsly stirred. Stirring was contin- ued for 20 min, and aft.er centrifugat.ion as in Step 2, the precipi- tate was discarded. To the supernatant (325 ml) was added 42.6 g of ammonium sulfate as before. The precipitate was collected by centrifugation, dissolved in 70 m of Buffer B (0.01 M potassium phosphate buffer pH 7.0, containing 10 mM 2-mercaptoethanol and 0.01 mM PLP) and dialyzed overnight against 3 liters of the same buffer. The volume of the dialyzed ammonium sulfate fraction was 79 ml.

Step 4: Acid Treatntent-Acetic acid (0.05 M) was added contin- uously to the dialyzed enzyme solution until the pH value fell to 5.1 as measured with the combination glass electrode (Corning). The solution was then allowed to stand undisturbed for 1 hour. After centrifugation at 18,000 rpm for 40 min (SS-34 rotor, Sorvall RC2-B), the phosphate concentration of the supernatant was raised to 0.05 M by the addition of 1 M potassium phosphate (pH 7.0) and the pH was adjusted to pH 7.0 by the addition of 0.1 N

NaOH. Step 5: Heat Treatnlent-The enzyme solution, contained in two

125.ml flasks, was heated with gentle swirling in a 65” water bath to a temperature of 5%GO”, andthen transferred to a 60-61” bath for an additional 10 min. After rapid cooling in ice water, the pre- cipitated protein was removed by centrifugation at 18,000 rpm for 60 min (83-34 rotor, Sorvall RC2-B) and the supernatant was frozen at -20”.

Step 6: DEAE-cellulose Column Chromatography-The heat- treated enzyme was thawed and centrifuged at 12,000 rpm for 20 min (GSA rotor, Sorvall RCB-B). The enzyme solution was then placed in a 50” water bath for 20 min. This treatment has been found to activate the enzyme and thus ehminate a shoulder on the main activity peak eluted from the DEAE-cellulose column. After chilling to 0”, NaCl was added to a final concentration of 0.05 M

and the solution was applied to a DEAE-cellulose column (2.6 X 60 cm) which had been equilibrated with Buffer C (0.05 M potas- sium phosphate buffer, pH 7.0, containing 10 mM 2-mercaptoetha- nol, 0.01 mM PLP, and 0.05 M NaCl). The enzyme solution (82 ml) was applied to the column at a flow rate of 2 ml/min and then eluted with a salt gradient which consisted of 650 ml of the equili- brating buffer in the mixing chamber and 650 ml of Buffer C con- taining 0.30 M NaCl in the feeding reservoir. Fractions of 15 ml were collected at a flow rate of 1.5 ml/min. The absorbance of each fraction at 280 nm was determined and even-numbered fractions were assayed for DAPA aminotransferase activity. Fractions 64 to 80 (250 ml), which contained 81% of the enzyme activity, were combined and the protein was precipitated by the addition of am- monium sulfate to 6070 saturation (98 g). The suspension was centrifuged at 12,000 rpm (GSA rotor, Sorvall RC2 B) for 1 hour The precipitated protein was dissolved in 10 ml of Buffer D (0.01 M potassium phosphate buffer, pH 8.0, containing 10m~ 2-mercapto- ethanol, 1 mM EDTA and 0.01 mM PLP) and dialyzed 5 hours against 2 liters of the same buffer. The dialyzed enzyme was cen-

trifuged at 18,000 rpm for 1 hour (SS-34 rotor, Sorvall RCB-B) and the supernatant was refrigerated overnight.

Step Y: Phosphocellulose Column Chromatography-The phos- phocellulose column (1.G X 20 cm) was equilibrated with 250 ml of Buffer D, the dialyzed enzyme solution (11.5 ml) from the DEAE cellulose column was applied, and eluted with starting buffer. Fractions of 3 ml were collected at a flow rate of 0.5 ml/min. Pre- liminary experiments showed the enzyme to be present in the non- adsorbed protein fraction. The nine fractions (27 ml) containing the nonadsorbed protein were made 0.02 M in phosphate by the addition of 0.5 M potassium phosphate (pH 6.8) and then concen- trated to 5.5 ml using a PM 30 membrane in a Diaflo ultrafiltra- tion cell (model 402) under nitrogen pressure (25 pounds).

Step 8: Hydroxylapatite Column Chromatography-The concen- trated eluate from the phosphocellulose column was then placed in a 50” water bath for 20 min as in Step 6. After chilling to 0”, the solution was applied to the hydroxylapatite column (3.2 X 4 cm) which had been equilibrated with 0.02 M potassium phosphate buffer, pI1 6.8, containing 10 mM 2-mercaptoethanol and 0.01 mM PLP. The column was washed first with 1 column volume (35 ml) of the equilibrating buffer, then with 1 column volume of the same buffer containing 0.05 M phosphate, followed by 1.5 column vol- umes of 0.10 M phosphate-buffer, and finally 1.5 column volumes of 0.50 M nhosnhate buffer. Fractions of about 3 ml were collected at a flow rate of 0.8 ml/min. The absorbance and the activity of each fraction was determined. The enzyme was eluted by 0.10 M phos- uhate buffer with Fractions 27 to 39 containing 87% of the total activity. The fractions were pooled (40.5 ml) then concentrated to 2.2 ml by ultrafiltration.

Steps 9 and 10: Sephadex G-100 Column Chromatography and Z-Mercaptoethanol Treatment-Two Sephadex G-100 columns (2.6 X 100 cm and 1.4 X 100 cm) were washed with Buffer C until the beds stabilized. The concentrated enzyme solution was made 10% in glycerol, applied to the larger column, and eluted with the start- ing buffer. Fractions of 4 ml were collected at a flow rate of 0.5 ml/min. Fractions 59 to 67 (35 ml) containing 74% of the enzyme activity were combined and concentrated to 1.4 ml by ultrafiltra- tion. 2-Mercapt.oethanol was added to a final concentration of 10% and the mixture incubated for 3Omin at 37”. The denatured protein was removed by centrifugation at 35,000 rpm for 30 min (40 rotor, Beckman model L).

Glycerol was added to the supernatant to a final concentration of 10% and the 2-mercaptoethanol, which interferes with the bio- assay for DAPA was removed by chromatography on the smaller Sephadex column. The enzyme was eluted from the column with Buffer C and fractions of 2.2 ml were collected at a flow rate of 0.2 ml/min. A single protein peak which coincided with the enzyme activity was observed. Each fraction was made 2O$Z& in glycerol and stored frozen at -20”.

RESULTS

Purification

A summary of the purification procedure is shown in Table

I which indicates an over-all purification of nearly 1099fold with

a 5% yield. After chromatography on the first Sephadex G-100

column, only two major bands are observed on polyacrylamide

disc gel electrophoresis as shown in Fig. 1A. After the second

Sephadex G-100 column only a single major protein band with

several minor components is seen. A gel electrophoretic pattern

of the peak fraction is shown in Fig. 1B. In order to estimate the

percentage purity of this fraction, the gel was scanned at a wave-

length of 550 nm using a linear transport attachment to the Gil-

ford Spectrophotometer (model 2400). The enzyme band consti-

tuted 86% of the total protein as estimated from the area under

the protein peaks on the scan.

That the enzyme activity coincided with the major protein

band observed on the gel (Fig. 1B) was demonstrated by slicing

the gel and assaying one half for activity, and staining the other

half for protein. In standard gels (7T0 run at pH 9.5), as well as

in two nonstandard gels, one of 15y0 run at pH 9.5 and one of 7%

run at pH 8.0, the peak activity was found in the slice containing

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TABLE I Purification of DAPA aminotransferase

1. Cell-free extract.. . 2. Protamine sulfate. . 3. (NH&SO,. 4. Acid treatment.. . 5. Heat treatment.. 6. DEAE-cellulose. 7. Phosphocellulose. . 8. Hydroxylapatite.. . 9. 1st Sephadex G-100..

10. 2nd Sephadex G-109.

-

.-

-

Volume

ml

244 300 79 98 90

250 33 40 32 13

I

-

Total protein

m

6560 4920

2730 1310 530 69 30 9.6 1.1 0.3:

-

_.

Specific Ihits’ activity6

x l@

a Micromoles of DAPA/min. * Micromoles of DAPA/mg of protein/min.

653 0.10

640 0.13 410 0.15

407 0.31

380 0.70 218 3.2 178 5.9 91 9.5 63 57 32 98

Yield

%

100

98 63 62 58 33 27 14 10

5

FIG. 1 (left), Polyacrylamide disc gel electrophoresis of DAPA aminotransferase. Electrophoresis of the enzyme solution (50 ~1) was for 1.25 hours at 3 ma/gel in a 4-cm standard gel (7%, pH 9.5) with a l-cm stacking gel, according to the method of Davis (17). The gels were fixed in 10% trichloroacetic acid for 1 hour, then stained with Coomassie blue (0.09%) and destained in 7oj0 acetic acid. A, fraction 63 of the first Sephadex G-100 column. B, Frac- tion 32 of the second Sephadex G-100 column (1.3 pg). The direc- tion of migration was from top to bottom.

FIG. 2 (right). Sodium dodecyl sulfate disc gel electrophoresis of DAPA aminotransferase. Fraction 33 of the second Sephadex G-100 column (50 ~1 containing 1.2 fig protein) was subjected to electrophoresis at 8 ma/gel for 4 hours.

the major protein band. This indicated that the major protein band seen on the disc gels was, in fact, DAPA aminotransferase.

Characterization of DAPA Aminotransjerase

hlolecular Weight Estimation by Gel Filtration on Sephadex G-%%--Gel filtration of DAPA aminotransferase which had been purified through the heat-treatment step was carried out on a Sephadex G-200 column in the presence of four marker proteins; y-globulins, yeast alcohol dehydrogenase, Escherichia coli alka- line phosphatase, and malate dehydrogenase. DAPA amino- transferase eluted from the column just before the alkaline phos-

phatase marker and from a plot of the elution volume of each marker protein uersu.s the logarithm of its molecular weight, the molecular weight of DAPA aminotransferase enzyme was esti- mated to be 84,000.

In a second gel filtration experiment, DAPA aminotransferase (purified through Step 9) was incubated 20 min at 37” with 4 mM Ado-Met in the presence of 0.12 M Tris-HCl (pH 8.5), in order to convert the enzyme to the readily resolved PMP form (see below). It was then chromatographed as before in the pres- ence of potassium phosphate buffer (0.10 M, pH 8.0) on the same Sephadex G-200 column, except that the markers run concur- rently with the enzyme were y-globulin, alcohol dehydrogenase, and ovalbumin. In this experiment the resolved DAPA amino- transferase eluted from the column just before ovalbumin. The elution volume corresponded to a molecular weight of 47,000. The DAPA aminotransferase recovered from the column repre- sented apoenzyme since it was inactive unless the fractions were assayed in the presence of PLP.

Molecular Weight Estimation by Sucrose Gradient Sedimenta- tion-DAPA aminotransferase, pretreated in two different ways, was sedimented in separate sucrose density gradients (5 to 20%). DAPA aminotransferase sedimented in an identical manner rela- tive to alcohol dehydrogenase regardless of whether the enzyme was preincubated with 7-keto-S-aminopelargonic acid and PLP or preincubated with Ado-Met. The latter treatment converted the enzyme to the PMP form and resulted in the dissociation of the cofactor from the enzyme as shown by the fact that the frac- tions from only this gradient were inactive when assayed in the absence of PLP. The sedimentation behavior of the enzyme which was not preincubated with either substrate was identical with that observed in the previous two gradients. Like the enzyme preincubated with 7-keto-8-aminopelargonic acid, it was active both in the presence and absence of PLP. From the sedimentation rate of DAPA aminotransferase relative to that of alcohol de- hydrogenase, an estimation of 104,000 daltons was obtained from these gradients.

Subunit Molecular Weight Estimation by Sodium Dodecyl Sul- fate Gel Electrophoresis-Sodium dodecyl sulfate gel electrophore- sis of DAPA aminotransferase after the final step in the purifi- cation revealed a single protein band, as shown in Fig. 2. This result indicates that if the subunits of the enzyme are noniden- tical, they are nonetheless of similar molecular weight and are not resolved by this technique. In order to obtain an accurate estimate of the molecular weight of the enzyme subunit, elec- trophoresis of the enzyme in triplicate sodium dodecyl sulfate gels was carried out concurrently with that of six standard pro- teins. From a plot of the average mobility of the standards against their molecular weight, it was determined that the average mo- bility of the enzyme subunit corresponded to a molecular weight of 47,000 f 3,000 indicating the presence of two subunits in the native enzyme.

Isoelectric Focusing of DAPA Aminotransjerase-The iso- electric point of DAPA aminotransferase as determined by iso- electric focusing was 4.7, but the recovery of activity was low due to the precipitation of the enzyme. Even with modification of the procedure, the maximum recovery attained was only 37% which was considered insufficient for the preparative use of this method.

Activity of Purified Enzyme with Mg2+-ATP ano? L-hlethionine- The ability of Mg*+-ATP and L-methionine to replace the Ado- Met in the crude cell-free extracts was previously ascribed to the presence of the Ado-Met synthetase enzyme. No activity could be detected with Mg2+-ATP and L-methionine in the puri-

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0 5 10 15 20 25 30

Time of Preincubation bin)

FIG. 3. Resolution of DAPA aminotransferase by preincuba- tion with Ado-Met in phosphate buffer. The enzyme was preincu- bated for the indicated times with (a) 4.2 mM Ado-Met (squares) (b) 0.017 mM 7-keto-Saminopelargonic acid (triangles); and (c) Hz0 (circles). The incubation was then continued for 30 min after the addition of the remaining substrate(s) with PLP (closed figures) and without PLP (open figures).

fied enzyme preparation, confirming our previous suggestion

(2). Cofactor Requirement-The stimulation of DAPA aminotrans-

ferase activity in dialyzed cell-free extracts by the addition of PLP (2, 22), suggested a requirement for this cofactor by the enzyme. It was found that the enzyme could be resolved by pre- incubation in the presence of Ado-Met and 0.125 M potassium phosphate buffer, pH 8.0, as indicated by the loss of activity when PLP was omitted from the incubation mixture (Fig. 3). In contrast to the effect of preincubation with Ado-Met, preincuba- tion with 7-keto-8-aminopelargonic acid or Hz0 had no effect upon the activity. Regardless of which substrate was present in the preincubation mixture when the remaining substrate was added in the presence of PLP, the same high level of activity was achieved, indicating that the resolution was rapidly and fully reversible. Resolution in the presence of Ado-Met and phosphate ions was nearly completein 10 min; the half-time was about 1.5 min. A low level of activity persisted in the absence of PLP be- yond 10 min of preincubation with Ado-Met; this was probably due to the low level of PLP carried over in the dialysis buffer.

Enzyme SpecijEty-Two keto analogs of 7-keto-8-amino- pelargonic acid, 8-keto-7-aminopelargonic acid and 7,8-diketo- pelargonic acid, can be transaminated by DAPA aminotransfer- ase to form DAPA (2, 23). However, the enzyme appears to be highly specific for Ado-Met as the amino donor. Of the Ado-Met analogs tested, neither S-adenosyl-L-ethionine, S-adenosyl-L- homocysteine. S-adenosyl- L- (2-hydroxy-4-methylthio)butyric acid, S-methyl-L-methionine, adenosine, nor methionine could support synthesis of detectable amounts of DAPA from 7-keto 8-aminopelargonic acid.

A much more sensitive test of the ability of an Ado-Met analog to donate an amino group to the enzyme is to test its ability to effect resolution of the enzyme. Since in this case the potential amino donor reacts with the enzyme in the absence of the other substrate, 7-keto-8-aminopelargonic acid, the latter substrate can exert no competitively inhibitory effect (see accompanying paper (23)). But more important, the potential amino donor need

react with the enzyme molecuIe only once in order to inactivate it for the subsequent assay carried out in the absence of PLP.

Several analogs of Ado-Met, various amino acids, and 8-keto- 7-aminopelargonic acid were all tested for their ability to effect resolution of the aminotransferase under the same conditions used with Ado-Met. A greater than 10% decrease in activity compared to the control level was considered to represent sig- nificant resolution of the cofactor from the enzyme. By this cri- terion, the following compounds were unable to effect significant resolution of the enzyme (the concentrations employed are given in parentheses) : S-adenosyl-L-homocysteine (4 mM), S-adenosyl- L-(2-hydroxy-4-methylthio)butyric acid (5 mM), S-methyl-n- methionine (4 mM), adenosine (4 mM), adenine (2 mM), L-methio- nine (4 mM), L-leucine (8 mM), and 8-keto-7-aminopelargonic acid (2 mM). The only compound, other than Ado-Met itself, which was found to effect the resolution of the enzyme was S- adenosyl-L-ethionine (4 mM). However, this resolution was very slow (only 50% in 30 min, compared to 90% in 10 min for Ado- Met) and may have been due to traces of Ado-Met in the com- mercial preparation of S-adenosyl-L-ethionine. In fact, a con- tamination of only 0.1% Ado-Met would explain the ability of this analog to resolve the enzyme very slowly since it was found that Ado-Met at a concentration of 4 JAM also showed about 50y0 resolution in 30 min.

Keto Product of Reaction-Although the amino product of the reaction, DAPA, had been extensively identified with the aid of very sensitive biological assay procedures (4), the expected keto product of Ado-Met, S- adenosyl- 2 - oxo- 4- methylthiobutyric acid, could not be detected. At the concentration levels attained in the enzymatic reactions, the use of chemical reagents for detec- tion were not sufficiently sensitive. We also had to consider the possibility that the keto product was unstable at pH 8.5, as similar sulfonium compounds have been reported to undergo decomposition with the formation of an unsaturated keto acid (24). A similar decomposition of the expected keto product would yield 5’-methylthioadenosine and 2-oxo-3-butenoic acid and the latter fragment should be readily separated from the former by passage of an acidified solution through a Dowex 50-H+ column. With the aid of S-adenosyl-L-[2-i4C]methionine, the stability of the keto product could be ascertained and any fragments containing the carbon 2 quantitated. Reaction mix- tures containing 1.25 mM of S-adenosyl-L-[2-i4C]methionine (2.9 x lo6 cpm) were incubated for various periods of time at 37”. In addition, a zero time control as well as controls with boiled enzyme and without 7-keto-8-aminopelargonic acid were in- cluded. After the incubation, all of the tubes were heated at 100’ for 1 min in order to promote the decomposition of the cr-keto product. After chilling to 0”, 20.~1 samples were removed for bioassay of DAPA. The l-ml reaction mixture was then acidified by the addition of 2 ml of 0.1 N HCl and applied to a 2-ml Dowex 50-H+ column. The column was washed with a 2-ml port.ion of 0.01 N HCl and the combined effluent and eluate (5 ml) was added to 18 ml of Aquasol scintillation fluid and counted. The column was immediately washed with a second portion of 0.01 N HCl (5 ml) and collected and counted in the same manner. The re- sults, presented in Table II, show that a nonenzymatic reaction was occurring as evidenced by the increased counts in both 40- min controls compared to the zero time control. On the assump- tion that the nonenzymatic reaction occurring during the 37” incubation was linear with time, the necessary correction was made at each time interval. The net counts thus obtained indi- cated the presence of 1.8 nmol, 3.0 nmol, and 3.7 nmol of the uncharged fragment after 10, 20, and 40 min, respectively. Bio-

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TABLE II TABLE III

Correlation of a deaminated derivative of Ado-Met with DAPA Effect of heating at 100° on stability of deaminated~derioative of

production Ado-Met

The reaction mixture contained in 1 ml of the following: 0.15 M

Tris Cl buffer (pH 8.5), 0.02 mM 7-Keto-8aminopelargonic acid,

1.25 mM [2J4C]Ado-Met, and 4.7 x 10e4 units of DAPA amino- transferase (Step 10). To obtain the enzymatic counts, the non- enzymatic controf reaction was assumed to be linear with time and the average of the net control counts was subtracted on that basis. The calculation of the nanomol of keto product is based

on a specific activity for the labeled Ado-Met of 2.32 X lo6 cpm/ pmol. Other details of the method are given in the text.

The reaction mixture contained in 1 ml of the following: 0.15 M

Tris Cl buffer (pH 8.5), 0.02 mM 7-keto-8-aminopelargonic acid, 1.25 mM [2-i4C]Ado-Met, and 4.7 X 1OV units of DAPA amino-

transferase (Step 10). To obtain the enzymatic counts, the non- enzymatic control reaction was assumed to be linear with time

and the average of the net control couuts was subtracted on that basis. The calculation of the nanomoles of keto product is based on a specific activity for the labeled Ado-Met of 2.32 x lo6 cpm/ pmol. Other details of the method are given in the text

Reaction

Zero time 10 min 20 min 40 min Control: 40 min (-7-

keto-%aminopelar- gonic acid)

Control : 40 min (boiled

enzyme)

-

GIOSS Enzy- counts matic

counts

7,410 12,600

16,400 20,100 11,500

11,400

5,206

8,900 12,700

4,100

4,OQfl

4,200 1.8 6,900 3.0 8,600 3.7

-

I Keto

noduct’ DAPA

nmol

1.7 2.9

3.9 0

0

(1 Postulated product, 2-oxo-3-butenoic acid (see Fig. 4).

assay of the amino product produced at these times indicated an equivalent amount of DAPA formed. This finding would sup- port the view that it is the 2-amino group of the methionine moiety of Ado-Met which is transferred to 7-keto-%aminopel- argonic acid in the DAPA aminotransferase reaction and that the product is unstable under the experimental conditions used.

It was of interest to determine whether the 2-keto derivative of Ado-Met would be sufficiently labile to decompose completely during the course of the reaction at 37” without the additional heating step. The reaction was run for 40 min with and without the I-min heating period with the appropriate controls. In addi- tion, a 20-min control (boiled enzyme) reaction was included to determine if the nonenzymatic reaction was indeed linear with time as had been assumed. The results are presented in Table III. It is clear that the omission of the heating reduces the zero time control value drastically (from 5790 cpm to 560 cpm) with- out reducing the net counts which represent the enzymatically- produced uncharged decomposition product. It can also be seen that the nonenzymatic reaction was approximately linear with time as assumed in the previous calculation. Since the decomposi- tion of the sulfonium ion of the product is therefore virtually

complete during the course of the reaction at 37” and does not require heating at 100” to bring it about, this experiment indi-

cates that the deaminated Ado-Met derivative produced in the

reaction is extremely labile in Tris-Cl buffer, pH 8.5.

DISCUSSION

On the basis of the experimental evidence presented above, the transamination of 7-keto-S-aminopelargonic acid by DAPA aminotransferase in the presence of Ado-Met can be formulated as shown in Fig. 4. The substrates for this enzyme are both un- usual for a transamination reaction. The biotin vitamer which is aminated in the reaction, 7-keto-8-aminopelargonic acid, is an a-amino ketone, rather than the usual cr-keto acid and, therefore, has the potentiality for acting as an amino donor as well as an amino acceptor. Ado-Met, which serves as the amino donor in

Reaction

A. Heated at 100” for 1 min

Zero time 40 min Control : 20 min (boiled

enzyme) Control : 40 min (boiled

enzyme) B. Not heated

Zero time 40 min Control : 20 min (boiled

enzyme) Control: 40 min (boiled

enzyme)

GIOSS counts

5,790 15,400

6,900

7,480

560

9,560 1,110

1,400

9,610

1,110

1,690

g,oocJ 460

840

7,940

8,160

Keto producta

nmol

3.4

3.5

*Postulated product, 2-oxo-3-butcnoic acid (see Fig. 4)

7-kato-S-amino- 7,8 dl*mino - polwgonic acid pelargonic acid

(‘I-KAP) @API)

x FWP-enzyme PLP-enzym.

S-sdenosyl-2-0x0-4 - mathylthiobutyric acid

Ado-Yet

nonenzymatic

2-oxo-3-butenoic acid + 5’-methylthioadenosine

FIG. 4. Proposed mechanism for the DAPA aminotransferase reaction.

the reaction, has not, to our knowledge, previously been reported to serve as a substrate of any transaminase. Ado Met is well known as the methyl donor in a wide variety of methylation

reactions (25), and after decarboxylation, as a propylamine donor

in spermidine and spermine biosynthesis (26). It has also been shown to be an activator of lysine-2,3-aminomutase, a PLP en- zyme (27).

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The amino product of 7-keto-8-aminopelargonic acid trans- amination was previously identified as DAPA by a comparison of its chromatographic and electrophoretic properties with those of an authentic standard (2). Its biological activity in feeding bioA mutants of Esche&hia co& provides the basis for a sensitive, simple, and direct bioassay procedure.

The unexpected finding that Ado-Met is required as the amino- donating substrate in the reaction has now been well established with the sucessful purification of the enzyme. The contaminants in the Ado-Met preparation (2) and the nonenzymatic decompo- sition products of Ado-Met in alkaline solution3 have been ruled out as amino donors.

Several lines of evidence suggest that Ado-Met serves as the source of the amino group. In the first place, the ability of Ado- Met to promote the rapid resolution of the enzyme indicates that it has the ability to donate an amino group to the enzyme-bound PLP. Such a transfer would break the covalent aldimine linkage to the enzyme, and permit the cofactor to dissociate from the enzyme in the presence of phosphate ions which compete for the phosphate binding site of the PLP molecule. A similar technique, but with much higher phosphate concentrations, has been used by Scardi et al. (38) and by Taylor and Jenkins (29) to resolve aspartate aminotransferase and leucine aminotransferase, re- spectively. Particularly significant is the inability of S-adenosyl- L-(2-hydroxy-4-methylthio)butyric acid to promote the resolu- tion of the enzyme, even though inhibition studies show it to bind more strongly and with greater specificity to the PLP form of the enzyme than any other analog tested (23). This suggests that the 2.amino group of the methionine moiety of Ado-Met is the one transferred to the enzyme-bound cofactor.

The possibility existed that a molecule of 7-keto-%amino- pelargonic acid could transaminate a second molecule to form one molecule of 7,%diaminopelargonic acid and one of 7,8- diketopelargonic acid with Ado-Met serving only to activate the enzyme. This alternative was discounted when it was found that 7,%diketopelargonic acid, a biotin vitamer which has no poten tial for self-transamination, could be converted to DAPA in the presence of Ado--Met and in the absence of 7.keto-&aminopel- argonic by the purified enzyme (23). In this reaction the source of the two amino groups transferred to 7,8-diketopelargonic acid must be Ado-Met.

Third, kinetic studies have established the involvement of Ado-Met as a substrate in a ping-pong mechanism in which 7- keto-8-aminopelargonic acid is a substrate inhibitor (23). This evidence that Ado-Met binds at the active center of one of two stable enzyme forms in a manner which is competitive with the inhibitory binding of 7-keto-8-aminopelargonic acid precludes a role for Ado-Met as a mere allosteric activator.

Fourth, if Ado-Met does indeed donate its 2-amino group to 7-keto-B-aminopelargonic acid, then the product of the reaction, S-adenosyl-2-oxo-4-methylthiobutyric acid, must appear in amounts equivalent to the DAPA synthesized. With the aid of S-adenosylL-[2-W]methionine as substrate we have found that the keto product readily decomposes under the experimental conditions used and a keto fragment containing C-2 of the me- thionine moiety accumulates in amounts equivalent to the DAPA produced. Certain cu-keto sulfonium compounds, of which the 2-keto derivative of Ado-Met is an example, have been shown by Bohme and Heller to decompose readily in alkali by the fol-

lowing mechanism (24) :

-OH 0 Hd II 11 ;:

XXC-CH-CH&+-R + X-C-CH=CH,

k + R’SR

Dimethylpropiothetin decomposes in alkaline solution to acrylic acid and dimethylsulfide (30), and an analogous decomposition of the 2-keto product of Ado-Met would yield 5’-methylthio- adenosine and 2-oxo-3-butenoic acid. The firm identification of the products of Ado-Met transamination would be aided by the availability of the authentic compounds. Neither S-adenosyl-2- oso-4-methylthiobutyric acid nor its decomposition product, 2-oxo&butenoic acid, have, to our knowledge, been synthesized.

The molecular weight estimates for DAPA aminotransferase obtained by two methods were 84,000 obtained by gel filt,ration, and 104,000 by sucrose gradient centrifugation. The average mass of 94,000 f 10,000 is taken as an estimate of the molecular weight of the holoenzyme. The highly purified apoenzyme eluted from the Sephadex G-200 column much later than did the holo- enzyme, at a position corresponding to a molecular weight of 47,000. This result suggested that resolution of the enzyme favors dissociation of the enzyme into subunits, as has been ob- served for aspartate aminotransferase (31, 32). The failure of the apoenzyme to dissociate on the sucrose gradient may have been due to the use of a partially purified enzyme preparation or to an effect of the sucrose itself. Sodium dodecyl sulfate disc gel electrophoresis established the molecular weight of the enzyme subunit as 47,000 f 3,000. The fact that the subunits show a single band on a sodium dodecyl sulfate gel indicates that the two subunits are of very similar molecular weights. Genetic evi- dence on this question has been provided by the studies of Cleary and Campbell who found a pattern of intragenic complementa- tion among 15 bioA mutants which suggested that bioA com-

prises a single cistron (33). Cleary and Campbell also point out that intragenic eomplementation can result from interaction between heterologous monomers in an enzyme normally com- posed of identical subunits. Thus the genetic evidence supports the finding that DAPA aminotransferase is multimeric and indi- cates that the subunits, shown by sodium dodecyl sulfate disc gel electrophoresis to be of similar molecular weight are, in fact, identical.

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G L Stoner and M A EisenbergPurification and properties of 7, 8-diaminopelargonic acid aminotransferase.

1975, 250:4029-4036.J. Biol. Chem. 

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