structure of 2-beto-3-deoxy-6-phosphogluconate aldolase · structure of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 246, No. 7, Issue of April 10, pp. 207.52083, 1971

Printed in U.S.A.

Structure of 2-Beto-3-deoxy-6-phosphogluconate Aldolase

II. CHEMICAL IWII~ENCE ICOR A THREE-SUBUNIT MOLECULE*

(Received for publication, August 27, 1970)

1). C. I~OBERTSON,~ 1~. H. HAMMERSTEDT,~ AND W. A. WOOD

From the Department o.f Biochemistry, Michigan State Lbriversity, East Lansing, Michigan &WY

SUMMARY

A three-subunit model is proposed to describe the subunit structure of Z-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase of Pseudomonas pufida. The following data are consistent with three identical or nearly identical subunits per 72,000. (a) A limiting molecular weight of 24,000 was calculated from the amino acid analysis data based upon the presence of 1 histidine residue. (b) The numbers of peptides detected with specific reagents in peptide-mapping experiments were consistent with three identical subunits. (c) Three moles of carboxy-terminal asparagine were released by digestion of S-carboxymethyl-KDPG aldolase with carboxypeptidase A. (d) Four radioactive peptides were observed after alkylation of the 12 cysteine residues with 1X!-iodoacetic acid, tryptic digestion, and column chromatography. (e) Three moles of 14C-pyruvate per mole of enzyme were bound by borohydride reduction. (f) A set of four hybrid species was detected by disc gel electrophoresis after dissociation and reassociation of mixtures of native and chemically modified KDPG aldolase. In addition, the molecular weight of the native aldolase determined by disc gel electrophoresis was in the range of 72,000 to 78,000, and the subunit molecular weight determined by disc gel electrophoresis in sodium dodecyl sulfate was 24,000 + 500.

Several unique features of 2-keto-3-deoxy-6phosphogluconate aldolase, including its stability, mechanism of catalysis, and distribution as described in the preceding paper (2), have prompted an investigation of the subunit structure of this aldolase. The chemical data presented herein show that the protein consists of three identical or nearly identical polypeptide chains, a conclu- sion also reached from physical measurements of molecular weights of the native molecule and its subunits (2).

* This work was supported by grant from the National In- stitutes of Health, and is Contribution 5209 of the Michigan Agri- cultural Experiment Station. A preliminary report of this work has been published (1).

1 Present address, Department of Microbiologv, Universitv of Kansas, Lawrence, Kansas 66044.

-” ,

$ Postdoctoral Fellow, National Institutes of Health. Present address, Department of Biochemistry, Pennsylvania State Uni- versity, University Park, Pennsylvania 16802.

EXPERIMENTAL PROCEDURE

Materials

Crystalline KDPGl aldolase was isolated as described in the preceding paper (2). The enzyme used in these studies showed one band by polyacrylamide gel electrophoresis, an Azso:AZGO ratio of 1.7 to 1.8, and a specific activity of 240 pmoles per min per mg.

The assay for KDPG aldolase has been described previously (3). Recently, it has been found that commercial preparations of lactic dehydrogenase are contaminated with cr-glycerol phosphate dehydrogenase and triose phosphate isomerase activities that convert one of the reaction products, n-glyceraldehyde&P, to a-glycerophosphate-P, with concomitant NADH oxidation, and give high activity values. Type III lactic dehydrogenase (No. 2625) obtained from Sigma is suitable for use in the coupled assay.

Trypsin, twice crystallized (TRL), carboxypeptidase A (COADFP), carboxypeptidase B (COBDFP), aldolase (rabbit muscle), pepsin (swine stomach), alkaline phosphatase (Es- cherichia co&), and lysozyme (egg white) were obtained from Worthington; pyruvate kinase (rabbit muscle) was from Boeh- ringer Jlannheim; carbonic anhydrase, chymotrypsinogen A, lactic dehydrogenase (type III), and cytochrome c were from Sigma; myoglobin (whale skeletal muscle), bovine serum albumin, and a-chymotrypsin were from Calbiochem. Lipoyl dehydrogenase was provided by Dr. John E. Wilson of this department.

L-l-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was prepared by the method of Kostka and Carpenter (4).

L-l-Tosylamido2phenylethyl chloromethyl ketone was purchased from Calbiochem. All other chemicals were reagent grade, available from commercial sources. Sephadex G-25 was purchased from Pharmacia. Iodoacetic acid-2-i4C (specific activity, 15.5 mCi per mmole) was purchased from Amersham-Searle Corporation, Des Plaines, Illinois. The iodoacetic acid-2-i4C (0.1 mCi) was mixed with 200 mg of unlabeled material and recrystallized from benzene-carbon tetrachloride (2 : 1, v/v). The final specific activity was 61,000 dpm per mg.

Methods

Amino Acid Analysis-Prior to hydrolysis, samples were de- salted on a column (0.6 X 12.0 cm) of Sephadex G-25. Aliquots

1 The abbreviations used are: KDPG, 2-keto-3-deoxy-6-phos- phogluconic acid; FDNB, I-fluoro-2,4-dinitrobenxene.

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2076 Skructure of d-Keto-3-deoxy-6-phosphogluconate Aldolase. II Vol. 246, No. 7

containing 0.5 to 1 .O mg of protein were transferred to hydrolysis vials and lyophilized. Ten milligrams of phenol were added to prevent destruction of tyrosine (5) followed by 0.5 ml of constant boiling HCl. The vials were degassed to remove traces of oxygen and evacuated to less than 50 p and sealed. After hydrolysis at 110” for 24 to 72 hours, excess HCl was removed on a rotary evaporator. The hydrolysate was dissolved in 0.2 M citrate buffer (pH 2.875) containing norleucine as an internal standard. An aliquot equivalent to about 25 pg of protein was applied to a noncommercial ultrasensitive amino acid analyzer.2

The area under each peak was determined with a planimeter. Variation among standard runs was +30/,. Half-cystine residues were determined either as cysteic acid (6) or as S-carboxymethylcysteine (7). Tryptophan was determined by the method of Edelhoch (8). Threonine and serine values were obtained by extrapolation to zero time hydrolysis.

Protein I>etermination-Readings at 280 and 260 rnp were used to calculate the protein concentration according to Warburg and Christian (9). The method of Lowry (10) was used with crystalline bovine serum albumin as a standard. Both methods were correlated with the dry weight obtained after heating at 105” until a constant weight was attained with correction for residual buffer. A ratio of 1 .O : 1 .O : 1.33 was obtained for the dry weight, Warburg-Christian, and Lowry values, respectively. The ex- tinction of a 1 .Os;. solution with an optical path of 1 cm, at 280 mp in 0.1 3. NaOH, was 8.63.

Peptide jlfa,pping-S-Carboxymethyl-KDPG aldolase was dissolved in 0.1 M NHJICO~ (5 mg per ml) and digested with L-l- tosylamido2phenylethyl chloromethyl ketone-trypsin (1: 100) at 37” for 4 hours. The reaction was stopped by lyophilization, the trgptic hydrolysate was dissolved in 0.1 M NH40H, and 20 pl(l.5 mg) were applied to Whatman No. 3MM chromatography paper. Descending chromatography was carried out in l-butanol-pyridine-glacial acetic acid-Hz0 (90 :60: 12 :72 v/v), for 20 hours. The high voltage electrophoresis was performed at pH 3.48 with pyridine-glacial acetic acid-Hz0 (1: 10 :89, v/v) and at 2500 volts for 75 min. Peptides were detected with 0.3% ninhydrin dissolved in acetone or with the cadmium-ninhydrin reagent described by Heilmann (11). Arginine was detected with the Sakaguchi stain or with phenanthrenequinone (12). Tyro- sine- and histidine-containing peptides were observed by staining with l’auly reagent (13).

Carboxypeplidase Digestion-Carboxypeptidase A (about 0.4 mg) was washed twice with 100 ~1 of ice-cold distilled water and dissolved in 100 ~1 of lO70 LiCl. Digestions were performed in 0.2 1\1 NH4HC03 in stoppered tubes at room temperature. The carboxypeptidase A to S-carboxymethyl-KDPG aldolase ratio was either 1: 5 or 1: 10. Aliquots were withdrawn at time intervals and frozen. The residual protein was removed by passage through a column (0.6 X 12.0 cm) of Sephadex G-25. Norleu- tine was added as an internal standard before the protein was removed to calculate recoveries. The fraction containing the amino acids released by carboxypeptidase digestion was taken to dryness with a rotary evaporator and dissolved in a minimal volume of pII 2.875 citrate buffer for analysis. Asparagine was determined as aspnrtic acid after hydrolysis with 2 N HCl for 2 hours at 100”. The excess HCl was removed and the residue was dissolved in pH 2.875 citrate buffer.

Preliminary experiments to establish optimal conditions for

2 1). C. Robertson, H. B. Brockmnn, W. I. Wood, and W. A. Wood, unpublished procedure.

carboxypeptidase A digestion with native KDPG aldolase showed that native enzyme in 0.2% sodium dodecyl sulfate or oxidized with performic acid give less satisfactory results than the method described above.

NHs-terminal Residue-The procedure used was a modification of that described by Morino and Snell (14). Fifteen milligrams of KDPG aldolase were dissolved in 3 ml of 6 M guanidine ($1 8.1) in 0.1 M NaHC03. Fluorodinitrohenzene (0.1 ml) was added, and the sample was incubated at 40” for 2 hours in the dark with continuous stirring. All subsequent manipulations were performed in reduced light. Excess reagent was removed by extracting the reaction mixture twice with 7 ml of ether. The dinitrophenylated protein was precipitated with 7 ml of 1 N HCl, collected by centrifugation, and washed twice with 3 ml of 1 N

HCl, 3 ml of ether, and 3 ml of acetone. The dinitrophenylated protein was dried overnight in a desiccator and dissolved in 12 N HCl. After transfer to the hydrolysis vial, an equal volurne of glass-distilled Hz0 was added. The sample was hydrolyzed for 16 hours at llO”, diluted to 1 N with respect to I-ICI, and extracted with ether. The ether extract was concentrated and subjected to descending paper chromatography on Whatman No. I paper. Isoamyl alcohol-l N NH40H (4 : 1, v/v) was used to separate the dinitrophenyl derivatives (15). Solvents used for thin layer chromatography of the dinitrophenyl derivatives were t-amyl alcohol-chloroform-glacial acetic acid (30 :70 :3, v/v) and benzene-pyridine-glacial acetic acid (80 :20 :2, v/v) (16).

Reaction of KDPG Aldolase with Iodoacetic Acid-%14C-Iodo- acetic acid-2J4C was used in the carboxymethylation procedure of Crestfield (7). Forty milligrams of KDPG aldolase were dialyzed against distilled water and lyophilized. The protein was dissolved in 3.75 ml of 8 M urea-Tris buffer (pH 8.6) containing 2 mg per ml of EDTA. Fifty microliters of freshly distilled 2- mercaptoethanol were added after purging with Nz for 20 min. After 4 hours, 135 mg of iodoacetic acid-2-14C, dissolved in 0.5 ml of 1 N NaOH, were added. The reactants were removed after 20 min by passage through a column (2.0 x 40.0 cm) of Sephadex G-25 that had been equilibrated with 0.02 M NH4HC03. Five- milliliter fractions were collected and the absorbance at 280 mp was measured. The fractions containing protein were pooled and lyophilized. The yield of S-carboxymethyl-KDPG aldolase was 80% or better in several experiments. The X-carboxymethyl-KDPG aldolase was dissolved in 0.1 M NH4HC03 (5 mg per ml) and digested with trypsin (1:50) for 4 hours at 37”. The reaction was stopped by freezing. Following lyophilization, the enzymatic digest was dissolved in 2 ml of 0.2 M pyridine acetate buffer (pH 2.1) and transferred quantitatively (including all of the precipitate) to the top of the Dowex 50-X2 column.

Peptide Chromatography-The methods described by Schroeder (17) were used for purification of the tryptic peptides. Dowex 50-X2 was washed with 2 N HCl, 2 N NaOH, 2 N pyridine, and 0.2 N pyridine acetate buffer, pH 3.1, before use. The gradient system consisted of 333 ml of 0.2 M pyridine acetate buffer, pH 3.1, and 666 ml of 2 sr pyridine acetate buffer, pH 5.0. The flow rate was 20 ml per hour and fractions of 1.5 ml were collected.

Every third tube was subjected to alkaline hydrolysis (18) and analyzed by the ninhydrin method of Yemm and Cocking (19). The peptides containing r4C were purified by high voltage electrophoresis at pH 3.5 and 6.5, located by radioautography, and eluted with 50y0 pyridine-Hz0 for amino acid analysis.

Cyanoyen Bromide Digestion-A 50-fold excess of cyanoaen bromide was added to S-carboxymethyl-KDPG aldolase that


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Issue of April 10, 1971 D. C. Robertson, R. H. Hammerstedt, and W. A. Wood 2077

had been lyophilized and dissolved in 70% formic acid (5 mg per ml). After 16 hours of incubation at room temperature, the reaction was stopped by dilution with 10 volumes of ice-cold water. Formic acid was removed either by repeated lyophilization or on a rotary evaporator, with care taken not to allow the temperature to rise above 35”. The digest was dissolved in 2 ml of 0.2 N

propionic acid for application to a Sephadex G-75 column (1 x

200 cm). The column was developed with 0.2 N propionic acid at 3.0 ml per hour with the collection of 1.5-ml fractions. Fol-

lowing alkaline hydrolysis (18), the fractions were analyzed by the method of Yemm and Cocking (19).

Substrate Binding-Since Silverstein a.nd Barker (20) have shown that sodium pyruvate-2.1% is stable in powder or fresh solutions, Na pyruvate-3-i4C (specific activity, 9.45 x lo5 dpm per pmole) was used in substrate binding experiments. The pyruvate in loo-fold molar excess was incubated with KDPG aldolase (5 to 15 mg per ml) in 0.1 M POn buffer, pH 6.0. A freshly prepared solution of NaBHI was added (loo-fold excess with respect to enzyme) in three aliquots over a 30-min period. The pH of the incubation mixture was adjusted to 6.0 with 2 M

acetic acid after each addition of borohydride. Ninety-nine per cent of the enzymatic activity was lost by this procedure. For

accurate determination of specific activity, recrystallized cold pyruvate was added to a freshly prepared solution of pyruvate-3- w. Then the pyruvate was completely reduced with lactic dehydrogenase and NADH and a replicate aliquot was counted directly. For determination of protein-bound radioactivity, the protein was separated from the low molecular weight material on a Sephadex G-25 column (0.6 x 12.0 cm). The recovery was better than 90%. The protein concentration was determined from the AZ80:A 260 ratio (Azso:AZGo = > 1.60) and replicate aliquots were counted with the xylene-dioxnne-cellosolve scintillation system of Bruno and Christian (21).

Preparation of Nybrids-Five milligrams of KDPG aldolase were maleylated with maleic-2, 3-14C nnhydride as described in the preceding paper (2). The maleylated aldolase had a specific

activity of 6.4 x lo5 cpm per mg and less than 0.50/ of its original enzymatic activity.

The dissociation and reassociation of either native or maleylated enzyme were performed according to the procedure of Deal (22). An aliquot containing 5 mg of protein was diluted to 1.0 ml and dialyzed overnight against 1000 volumes of 0.03 M potassium phosphate, pH 6.0. Five microliters of P-mercaptoethanol and 900 mg of urea were added to effect dissociation (final volume, 1.7 ml). Less than lo/;! of the initial enzymatic activity was detected after incubation at room temperature for 45 min with stirring.

For reassociation of the native and maleylated proteins, aliquots (0.32.ml) were withdrawn from the dissociation solvents and diluted 100.fold with ice-cold potassium phosphate, pH 8.0. After 1 min, the solutions were placed in a water bath at 20” for 30 min. The hybrid set was prepared by combining the remainder of the two solutions. After thorough mixing, the solution was diluted as described above. Sixty per cent of the enzymatic activity was recovered after dissociation and reassociation of the native enzyme. The recovery of enzymatic activity

in the hybrid mixture was 560/(, of that expected from the native protein. Each solution was dialyzed against 10 volumes of 0.02 M potassium phosphate buffer, pH 8.0, with three changes of

dialysate. The solutions were each concentrated with a Diaflo mem-

brane to approximately 1 ml and centrifuged to remove turbidity. The recovery of enzymatic activity and i4C in the concentration step was 55 to 65%.

The samples were subjected to disc gel electrophoresis as described by Davis (23). Each tube contained 2.0 ml of 7.5% acrylamide, 0.1 ml of stacking gel, and sample gel. The samples

were electrophoresed until the tracking dye was I to 2 cm from the end of the gel. Duplicate gels were run and, after removal from the tubes, the gels were either stained with Coomassie blue or cut into 2-mm slices. Each slice was extracted overnight at 4” in 0.2 ml of 0.05 bf potassium phosphate, pH 7.4. An aliquot (1 to 10 ~1) was removed for determination of KDPG nldolase activity and the remainder of the gel and buffer was transferred, with rinsing, to a scintillation vial for determination of radioactivity. The companion gel, after staining with Coomassie blue, was scanned at 570 mp in a Gilford spectrophotometer equipped with a linear transport attachment.

The molecular weight of KDPG aldolase was determined by the gel electrophoresis method of Hedrick and Smith (24). Mi- grations of standards at gel concentrations of 12, IO, 8, and 6% were used to calculate the slope-molecular weight relationship. The gels were 10 cm in length with a diameter of 5 mm. The running time was 75 min, and the dye front was marked by piercing the gel with a section of 24-gauge copl)er wire. The staining solution was prepared by dissolving I .25 g of Coomassie blue in a mixture of 454 ml of 50% methanol and 46 ml of glacial acetic acid. The solution was filtered before use. The gels were destained with several changes of destaining solution (75 ml of glacial acetic acid, 50 ml of methanol, and 875 ml of water) over a period of 24 hours (25).

Electrophoresis in the presence of sodium dodecyl sulfate was performed as described by Weber and Osborn (25). The gels were destained with several changes of destaining solutions over a period of 18 to 24 hours. There was sufhcient contrast for photography within 48 hours. The relative mobility of each protein was calculated by dividing the migration distance by that of an internal standard, myoglobin.

RESULTS

Amino Acid Analysis-The amino acid analysis of 25 pg of KDPG aldolase is shown in Table I. The values for 24 and 70 hours of hydrolysis arc expressed as residues per 9 tyrosine residues to correct for slight differences in the amounts of protein hydrolyzed. The number of residues per mole of aldolase is derived from these values. The weight of each amino acid obtained by integration is given in the last column. The recovery

was 24.82 pg of 25.00 pg hydrolyzed, or 99%. The good recovery supports the validity of the calibration procedures used and shows that the aldolase contains negligible carbohydrate or other ninhydrin-negative material. Considering the reproducibility of the amino acid analyzer to be +39:,, the residues present in small amounts, except methionine, are divisible by 3. Any methionine converted to the sulfone during the hydrolysis would not have been detected since methionine sulfone elutes from the analyzer column coincident with aspartic acid. Consequently, the rnethi- onine value is probably low.

Calculations based upon 9 tyrosine, I2 cysteine, 21 lysine, and 66 glycine residues yielded similar results. A limiting molecular weight of 24,000 was calculated assuming 1 histidine residue per subunit. This value is identical with that obtained for the subunit by physical methods (2).


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2078 Xtructure of 2-Keto-3.deoxy-6-phosphogluconate Aldolase. II Vol. 246, No. 7

TABLE I TABLE II

Amino acid analysis of KDPG aldolase

The data were obtained from the average of duplicate analyses of 0.5 mg each of KDPG aldolase hydrolyzed for 24 and 70 hours with 0.5 ml of constant boiling HCI in sealed, evacuated tubes at 110”. Twenty-five micrograms were applied to an ultrasensitive

Summary of peptide-mapping experiments

All conditions were as described in Fig. 1.

Detection reagent

Theoretical

Observed

3 subunits 4 subunits

residues residues

22 16 21-23 15 11 12-13 3 2 3 1 1 3 2 3

analyzer.

Amino acid

Cysb (Cma) ...................... Asp ............................. Thrc. ............................ Serc ............................. Glu ............................. Pro ............................. Gly ............................. Ala .............................. Val.............................. Met ............................. Ile .............................. I,eu ............................. Tyr ............................. Phe ............................. Lys ............................. His .............................. Arg ............................. Tryd ............................ NHae ............................

Sum of weights Correction for water addition in

hydrolyses Corrected weight in terms of un-

hydrolyzed sample -

Residues Per 9 tyrosine residues

24 hrs 70 hrs

12.32 50.17 29.94 25.86 61.07 47.28 61.53 92.51 39.67 20.56 52.86 60.34 9.00

19.78 21.36

3.16 43.94

50.58 28.59 23.01 59.85

65.85 92.40 44.01 19.71 55.32 65.46 9.00

21.87 21.36

3.00 46.50

12 50 31 25 60 47 66 92 44 20 55 65 9

22 21

3 45 11 10

1

fig 0.72b 2.24 1.23 1.02 2.93 1.92b 1.65 2.74 1.72 0.98 2.42 2.86 0.54 1.20 1.04 0.16 2.70 0.01 0.17

28.85 -4.03

24.82

a Cm, carboxymethyl. Determined from a carboxymethylated preparation of KDPG aldolase.

b Calculated from 24.hour analysis. c Extrapolated to zero hour hydrolysis. d Determined by spectral methods described under “Materials

and Met,hods.” e Ten amide residues were assumed.

The 12 half+ystines/72,000 daltons were obtained either as cysteic acid or S-carboxymethylcysteine. Twelve sulfhydryl residues were also titrated with Elman’s reagent with urea-denatured KDPG aldolase in the absence of reducing agent9; thus, KDPG aldolase contains no disulfide bonds. Eleven tryptophan residues were found by Edelhoch’s procedure (8). A small number of amide bonds was indicated by the relatively low p1 of 4.8 (2). The moles of asparagine and glutamine were assumed to be 10 for the purposes of these calculations.

Peptide-mapping Experiments-The number of identical subunits and evidence for nonidentical subunits can be estimated from peptide maps of tryptic digests. Since the aldolase contains 66 arginine plus lysine residues, it is possible to calculate

the number of peptides for three or four identical subunits as shown in Table II. Although interpretation of peptide maps is tenuous at best, it has been possible to observe repeatedly 21 to

3 K. A. Decker, H. MGhler, and W. A. Wood, manuscript in preparation.

Ninhydrin (or-amino) . Phenanthrenequinone (arginine) Pauly (tyrosine). Pauly (histidine). Ultraviolet light (tryptophan).

+

+--- HVE pH 3.5

FIG. 1. Schematic of peptide map prepared from the tryptic digestion of S-carboxymethylated KDPG aldolase. The tryptic hydrolysate was dissolved in 0.1 N NH,OH and 20 ~1 (1.5 mg) were applied at the origin of each sheet of Whatman No. 3MM chromatography paper. Descending chromatography in l-butanol-pyridine-glacial acetic acid-Hz0 (90:60:12:72, v/v) was followed by high voltage electrophoresis at pH 3.48 as described under “Ex- perimental Procedure.” The circled areas denote ninhydrin color and the shaded areas identify coincident staining for arginine. The areas marked P react with Pauly reagent for tyrosine and the spots designated C2MS contain radioactivity when XXodo- acetic acid was used in preparing the carboxymethyl derivative.

23 ninhydrin-positive spots of which 12 to 13 gave a response to reagents for arginine (Fig. 1). Three peptides (marked P in Fig. 1) contained tyrosine and one contained histidine, as revealed with Pauly reagent. Three peptides were observed by ultraviolet light indicative of tryptophan (Table II). The spots de- noted CMS contained X-carboxymethyl-cysteine and were detected by radioautography of KDPG aldolase that had been alkylated with Y-iodoacetic acid, digested with trypsin, and subjected to peptide mapping. These results are very close to the values expected for three identical subunits and are difficult to reconcile with the expected values for four subunits.

COOH-terminal Residue-The carboxy-terminal residue was determined by digestion of S-carboxymethyl-KDPG aldolase with carboxypeptidase A. The rate of release of amino acids is


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7-

6-

, /

/’ 0’

/ ,

/’

,/‘Alonine

/’ Asporogine /’

;rr.,. ,/‘..

...,~~‘.T&L/~;~~~O

‘1

.****. . . . . ...****** “-4yz *....... *..-.

*... . . . . . . . .** lsoleocine

I I I I I 0 12 24 36 40 60 72 04 96

Time (Hours)

FIG. 2. Carboxypeptidase A digestion of S-carboxymethyl- KDPG aldolase. S-Carboxymethyl-KDPG-aldolase was digested with carboxypeptidase A (1:lO) in 0.1 M NHdHCOa at 25” with stoppered tubes. Aliquots were removed at various time intervals and frozen. The protein was removed with a column (0.6 X 12.0 cm) of Sephadex G-25. Asparagine was determined as aspartic acid after hydrolysis with 2 N HCl for 2 hours at 100”.

shown in Fig. 2. The values for asparagine and leucine reached a maximum at 3 moles per mole of aldolase. The release of lysine followed a similar pattern; however, the release of alanine con- tinued linearly for 96 hours. The long incubations were required because of the rate-limiting release of asparagine.

If leucine had been the carboxy-terminal residue, large amounts of leucine and much smaller amounts of asparagine would have been expected at earlier time intervals since it is known that leu- tine is released at a faster rate than asparagine. It should be noted that asparagine was determined as aspartic acid after removal of the protein and hydrolysis with 2 N HCl. Controls included a similar sample not subjected to acid hydrolysis to correct for residual aspartic acid and a blank in which the substrate was omitted to measure the free amino acids released by the autodi- gestion of carboxypeptidase A. In addition, use of X-carboxymethyl-KDPG aldolase provided a denatured substrate for optimal digestion by carboxypeptidase A.

NH2-terminal Residue-The NHz-terminal residue was identi- fied by dinitrophenylation of the aldolase, hydrolysis, and chromatography as described under “Experimental Procedure.” As shown in Table III, only one dinitrophenyl amino acid was found on chromatograms of the ether-soluble fraction. In four solvent systems, the unknown had an RF value identical with that of dinitrophenyl-threonine. It is of interest to note that only e-dinitrophenyl-lysine was observed unless the protein was denatured in 6 M guanidine hydrochloride before addition of FDNB.

X-Carboxymethylcysteine-containing Peptides-The cysteine residues of KDPG aldolase were alkylated tith iodoacetic acid- VC, as described under “Experimental Procedure.” The S- carboxymethyl derivative was digested with trypsin and applied to a Dowex 50-X2 column with the results shown in Fig. 3. Based upon 12 cysteine residues and the fact that KDPG aldolase contains no disulfide bonds, three radioactive peptides would be erpected from four identical subunits and four radioactive

TABLE III Identijkation of NHS-terminal threonine

Fifteen milligrams of KDPG aldolase in 3 ml of 6 M guanidine-1 M NaHC03 (pH 8.1) were incubated with 0.1 ml of fluorodinitro- benzene at 40” for 2 hours with continuous stirring. Excess reagent was removed as described under “Materials and Methods.” The dinitrophenylated protein was hydrolyzed with 6 N HCI for 16 hours at 110’. The hydrolysate was diluted to 1 N with respect to HCI and extracted with ether. The ether extract was concentrated to dryness and subjected to chromatography.

Solvent

Paper chromatography Isoamyl alcohol-l M NH&OH.. . . _. .

Thin layer chromatography Tert-amyl alcohol-chloroform-acetic acid

(30:70:5, v/v).. Benzene-pyridine-acetic acid (80:20:2, v/v).

Polyamide layers I-Butanol-acetic acid (90:20, v/v). . . .

RF unknown:

0.52 0.51

0.12 0.12 0.18 0.18

0.42 0.42

Dini- rophenyl- .hreonine

Fraction Number

FIG. 3. Elution of the tryptic peptides of S-carboxymethyl- KDPG aldolase on a column (0.9 X 100 cm) of Dowex 50-X2. Forty milligrams of KDPG aldolase were alkylated with iodoacetic acid-Z-W as described under “Experimental Procedure.” The tryptic digestion was stopped by lyophilization, dissolved in 2 ml of 0.2 M pyridine acetate buffer (pH 2.1), and transferred to the peptide column. The conditions for chromatography and analysis are described under “Experimental Procedure.”

peptides from three identical subunits. These results are possible only if a residue susceptible to tryptic cleavage, either lysine or arginine, is found between the cysteines distributed throughout the polypeptide chain. If this is the case, a 1 :l ratio of either lysine or arginine to X-carboxymethylcysteine will be observed.

Four radi,\active peptides, T-l, T-3, T-4, and T-5, and a small amount of undigested material eluted with 2 M pyridine were detected. The 14C recovery was 80% and 65% in two experiments. The low recovery was probably due to the fact that thiodiglycol was not included in the buffers and that some oxidation of X-carboxymethylcysteine to the sulfone occurred.

The W-S-carboxymethylcysteinyl-peptides were purified by high voltage electrophoresis as described under “Experimental Procedure” and subjected to amino acid analysis. The results


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2080 Structure of 2-Keto-S-deoxy-6-phosphogluconate Aldolase. II Voi. 246, No. 7

TABLE IV TABLE V

Characterization of 14C-carboxymethylcysteinyl peptides Binding of ‘4C-pyruvate by borohydrirle reduction

Peptides T-l, ‘T-3, and T-4 shown in Fig. 4 were purified by high voltage electrophoresis at pH 3.5. T-5 was purified by high voltage electrophoresis at pH 6.5. The radioactive peptides were detected by radioautography and eluted with 50% pyridine-H?O. The W-S-carboxymethylcysteinyl peptides were hydrolyzed with constant boiling HCl by the conditions described under “Mate- rials and Methods.” After removal of the excess HCl, the hydrolysate was applied to an ultrasensitive amino acid analyzer. Values are reported in nanomoles. The values in parentheses represent assumed number of residues.

KDPG aldolase (0.94 to 3.76 mg) was incubated for 20 min in 0.4 ml of 0.05 M phosphate buffer, pH 6.0, with a loo-fold excess of pyruvate-3-14C (specific activity, 945 dpm per nmolc). A lOO- fold excess of sodium borohydride was added in three aliquots over a 30-min time period. The pH was adjusted to 6.0 with 2 N acetic acid after each addition of borohydride. The reactants were removed by passage of a 0.2.ml aliquot of the reactsion mixture through a Sephadex G-25 column (0.6 X 12.0 cm). The protein concentration was measured by ~4~sa:~42~0. The radioactivity was determined with the xylene-dioxane-cellosolve (1:3:3, v/v) scintillation mixture of Bruno and Chrisliar (18) containing 47, Cab-0-Sil. Ninet,y-nine per cellt of the enzymatic activity was lost by this procedllre. Amino acid

Cys (Cm(a) ..... Asp ........... Thr ........... Ser ......... Gill ......... Pro. ............ Gly ............ Ala ......... Val. ........... Met ............. Ile ........... LeLl ......... Tyr ............. Phe ............. Lys ............ His. ............ Arg .............

T-l T-3

0.69 (1) 2.08 (2)

1.14 (1) 2.77 (3)

9.08 (1) 9.28 (1)

10.27 (1)

5.76 (G) 10.06 (1)

2.18 (2) 2.24 (2)

18.62 (2)

Amino acid content

9.48 (1)

-7

T-4

7.08 (1) 14.89 (2) 6.97 (1)

15.49 (2) 8.84 (1)

16.88 (2)

T-5

9.58 (1) 11.36 (1) 7.96 (1)

11.19 (1)

14.63 (1) 12.40 (1) 12.27 (1)

11.82 (1)

G.53 (1)

7.14 (1) 10.60 (1)

Q Cm, carboxymelhpl.

in Table IV show that Peptide T-l did not contain arginine or lysine. However, T-l contained 1 mole of serine, 3 moles of glutamic acid, and 6 moles of alanine; thus, the composition is unique and not the result of degradation of one of the other 14C- S-carboxymethylcysteine-containing peptides. It is evident that a tryptic peptide derived from the COOH terminus of the polypeptide chain would not contain lysine or arginine. Thus, T-l probably arose from the COO&terminal region of KDPG-aldolase. The other radioactive peptides contained 1 residue each of S-carboxymethylcysteine and either lysine or arginine.

The fact that lysine was detected in the COOH-terminal region during carboxypcptidase digestion whereas isolation of a terminal 14C-X-carboxymethy1cysteinyl-peptide of 17 residues (T-l) did not contain lysine seems to be inconsistent (Fig, 3 and Table IV). Thus, it is likely that T-l was part of a larger COOH-terminal fragment and was formed as a result of chymotryptic-like activity in the trypsin preparation (26). Removal of a small COOH- terminal peptide containing the lysine residue would increase the acidity of the residual peptide which correlated with the elution position of T-3. (Fig. 3).

Cyanogen Bromide Digestion-Since KDPG aldolase contains a limited number of methionine residues, it was expected that the number of subunits could be determined from the number of peptides produced by cyanogen bromide digestion of S-carboxymethyl-KDPG aldolase. S-Carboxymethyl-KDPG aldolase was digested with CNBr as described under “Experimental Pro- cedure.” The digest was dissolved in 0.2 M propionic acid and

Concentration of KDPG aldolase “C-Pyruvate bound

m&T/ml moles/mole of aldolase

2.35 2.6 4.7 2.9 9.4 2.7

applied to a Sephadex G-75 column (1 x 200 cm). The frag- ments were similar in size and could not be resolved into more than four poorly separated fractions. Thus, no useful data on the subunit structure of KDPG aldolase resulted from this approach.

Reductive Binding of Pyruvate with Borohydride-The moles of substrate bound per mole of enzyme are an indication of the subunit composition if each subunit contains a substrate-binding site. KDPG aldolase has been shown to form a Schiff base complex with pyruvate that can be stabilized with borohydride (27, 28). Following 20-min incubation with a loo-fold excess of pyruvate at three different protein concentrations, KDPG aldolase was reduced with borohydride as described under “Experi- mental Procedure.” As shown in Table V, approsimatcly 3 moles of 14C-pyruvate were bound in each case. Greater than 99% of the enzymatic activity was lost by this procedure. These data are consistent with three active sites.

Hybridization ,%periments-Because most of the methods to determine the number of polypeptide chains per oligomer yield minimal values, an approach not dependent on a knowledge of the molecular weight and amount of protein was desirable. Hy- bridization of two forms of an oligomeric protein in vitro produces a characteristic number of hybrid species. For example, a five- membered hybrid set is observed after dissociation and renssocia- tion of binary mixtures of fructose-l, 6-I’*-aldolase isolated from various tissues (29). These data provided strong evidence that the protein is composed of four polypeptide chains.

Multiple forms of KDPG aldolase have not been observed; therefore, a second form for hybridization was produced by chemical modification. It was reported in the previous paper that maleic anhydride does not affect dissociation of KDPG aldolase; that is, by high speed equilibrium analysis the molecular weight was 72,500 f 500 (2). Mnleylated KDPG aldolase was cn- zymatically inactive, yet the protein could be reconstituted with the dissociation and reassociation conditions described under “Experimental Procedure.” Thus, KDPG aldolase, chemically modified with maleic anhydride, was a suitable variant for the

following hybridization studies. While this paper was in preparation, Meighen and Schachman (30) described a method with


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Issue of April 10, 1971 D. C. Robertson, R. H. Hammerstedt, and W. A. Wood

01 I I I I 2 4 6 8

Centimeters

FIG. 4. Controls for the hybridization of native and maleylated KDPG aldolase. Native and chemically modified a.ldolase was dissociated and reassociat.ed with the conditions described under “Experimental Procedure.” Gel B was native aldolase, dissociated and reassociated; Gel 13 was maleylated aldolase; and Gel C was observed after dissociation and reassociation of equal amounts of native and maleylated aldolase.

succinic anhydride to form a chemically modified variant of fructose-1 , 6-Pz aldolase.

The reaction with maleic anhydride affects a change in the net charge from +1 to -I for each modified residue. The variation in net charge of (+21 to -21) suggested that the species could be readily separated with disc gel electrophoresis. Native and maleylated KDPG aldolase, when electrophoresed individually or together, migrated as single components of RF (0.44 and RF 0.77, respectively. Similarly, upon dissociation in urea folloaTed by reassociation by the procedure described under “Methods,” electrophoresis individually or together yielded the same two major peaks of the RF values reported above. In addition, there was one minor, very rapidly migrating peak which coincided with the dye front. These data, shown in part in Fig. 4, A and B, are for runs wherein the dye front was allowed to exit from the gel and, therefore, do not show the fast moving minor peak. Partic- ularly important is the fact that no species intermediate between the two major peaks was detected when the native or chemically modified proteins were dissociated and reassociated individually before electrophoresis. The identical migration of the dissociated forms with the nondissociated forms, as well as the determination of the molecular weights of the native and maleylated forms as 72,000 (2), establishes the electrophoretic limits of the hybrid set. Fig. 4C shows that four peaks at RF 0.44, 0.55,0.66, and 0.77 were observed. Hence, the two new hybrid peaks only arise by mixing the dissociated native and maleylated forms before reassociation.

A typical experiment with 14C-maleic anhydride is shown in Fig. 5. Equal amounts of native and chemically modified KDPG aldolase were dissociated, combined, and reassociated as described under “Experimental Procedure.” Five peaks designated I to V were detected either by absorbance scans at 280 mp or in gels

Fraction Number

FIG. 5. Electrophoretic separation of the hybrids of KDPG aldolase. Maleylated and native KDPG aldolase were dissociated in urea, reassociated, and concentrated as described under “Ex- perimental Procedure.” Gel electrophoresis at pH 8.3 gave a distribution consistent with the following assignment: I = Ns, II = N2M, III = NMs, IV = Ma, and IJ = M (and probably N), where N is a native subunit and M is a maleylated subunit polypeptide chain.

stained with Coomassie blue. Companion gels were cut into 2-cm slices and extracted to determine radioactivity and enzymatic activity. Peaks I to III showed enzymatic activity, whereas II to V contained radioactivity. As noted above, Peak V migrated with the tracking dye and probably represents a single polypeptide chain.

The data in Fig 5 and the experiments shown in Fig. 4, A to C, show that Peak I represents the native protein, Peak IV is the chemically modified form, and the intermediate Peaks II and III are hybrid species containing both radioactivity and enzymatic activity. The four-membered hybrid set would be expected from a protein composed of three polypeptide chains.

Complete dissociation of native KDPG aldolase in 8 M urea has been established by optical rotary dispersion measurements and calculation of zero or-helix content from 50 and Hbo. In sucrose gradients, the material was converted to a much slower sedimenting form. In addition, all -SH groups become available for titration with Elman’s reagent, whereas in the native aldolase only half of the 12 -SH groups react.3 There are no disulfide bonds in the native molecule. It was also shown that reassociation produces active aldolase determined to be identical with the native form from optical rotary dispersion measurements, crystal form, specific activity, kinetics of titration with Elman’s reagent, and density gradient centrifugation.3

From the areas under the peaks averaged from the two electrophoretic runs, the relative abundance of the forms Na, N2M, NM2, Ma (where N is a native subunit and M is a maleylated subunit polypeptide chain) was roughly 8 :4 :2 : 1. These values do not fit well a cubic equation generated from (a~ + by)3 for any values of a and 6, nor should this be expected considering the complex nature of the association process. However, it is evident that the maleylated form does not associate as readily as the native enzyme, perhaps by a factor of 2. Thus, it may be rea- soned that since the native enzyme dissociates in 8 M urea, the maleylated form also completely dissociates.

These data show that both the native and maleylated aldolases yielded three other species, the two intermediate forms and the


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2082 Structure of SKeto-S-deoxy-6-phosphogluconate Aldolase. II Vol. 246, p\‘o. 7

2.c

E g 1.5

I .c IL 5

I I 1 , IO 15 20

Molecular Weight x 10e4

FIG. 6. Determination of the molecular weight of KDPG aldolase by disc gel electrophoresis. The slope characteristic of each protein was obtained with gel concentrations of 12,10,8, and 60/,. The proteins were dissolved in 0.02 M potassium phosphate, pH 6.0, and applied to the gel with 10% sucrose. The bands were located by staining with Coomassie blue and the dye front was marked with a section of 24-gauge copper wire.

6

+- b4 - x i g 3-

3

G s JJ? 2- 3

o Chymotrypsinogen A ‘A KDPG 0

Trypsin

\

Aidolose

o Myoglobin

\ 0,Lysozyme

Cy&hrome c

I I I I I I I 02 0.4 0.6 0.8 I 0 1.2

Relative Mobility

FIG. 7. Determination of the subunit molecular weight of KDPG aldolase with 10% polyacrylamide gels. The relative mobility of each polypeptide chain was calculated relative to an internal standard, myoglobin. About 10 pg of each protein were applied to gels after dissociation in 0.02 M sodium phosphate, pH 7.0, containing 1% sodium dodecyl sulfate and 1% mercaptoethanol.

rapidly migrating intact subunit, again showing that dissociation occurred. From the data in Fig. 4, A and B, it is evident that these intermediate forms are not produced upon dissociation and reassociation individually, and, hence, are not partially dissociated forms.

Determination of Molecular Weights by Disc Gel Electrophoresis -Even though all the data presented in this and the previous paper have been consistent with three identical subunits, it was considered desirable to obtain additional molecular weight data

of the parent and subunit by methods which did not depend on assumptions involved in ultracentrifugation. This was especially important since the amino acid analysis data, peptide-mapping experiments, and moles of COOH-terminal residues released by carboxypeptidase, as well as moles of pyruvate bound by borohydride reduction, were calculated based on a molecular weight of 72,000.

The experiments to determine the molecular weight of native KDPG aldolase by gel electrophoresis by the procedure of Hed- rick and Smith (24) are summarized in Fig. 6. Four different gel concentrations (12, 10, 8, and 6s1) were used to calculate the slope characteristic of each protein. In three experiments, molecular weights of 72,000, 75,000, and 78,000 were observed.

The results of electrophoresis in the presence of sodium dodecyl sulfate are shown in Fig. 7. During seven runs, a molecular weight range of 23,500 to 24,750 was observed. Trypsin con- sistently exhibited a molecular weight of 25,000; however, this value is within the margin of error found by others (25,31). The data support a subunit molecular weight of 24,000 % 2,000 reported in the previous paper (2).

DISCUSSION

Chemical evidence reported herein and the physical evidence presented in the previous paper (2) strongly support the presence of three identical or nearly identical subunits in KDPG aldolase. Two independent lines of evidence, ultracentrifugation and a method utilizing disc gel electrophoresis, indicated a molecular weight of 72,000 for native KDPG aldolase. In addition, three different approaches indicated a subunit molecular weight of 24,000. (a) A limiting molecular weight of 24,000 was calculated from amino acid analysis data based on the presence of 1 histidine residue; (b) a molecular weight of 24,000 + 2,000 was observed by ultracentrifugation in guanidine hydrochloride-O.2 M mercaptoethanol; and (c) a subunit molecular weight of 24,000 f 500 was determined with disc gel electrophoresis in the presence of sodium dodecyl sulfate.

The fact that 3 histidine residues/72,000 were found also is consistent only with a three-subunit model. In view of the complete recovery of the weight of samples subjected to amino acid analysis, which supported the color constants for each amino acid, it is unlikely that the histidine value is low by 250/,.

The use of specific reagents for the detection of peptides containing arginine, tyrosine, histidine, and tryptophan, in addition to ninhydrin staining, increases the value of peptide maps. The numbers of peptides expected from a protein with three identical subunits were observed both in peptide maps of tryptic digests and by column chromatography of radioactive peptides after alkylation with W-iodoacetic acid; however, methods based upon specific fragmentation techniques and analysis of the numbers of peptides are likely to be less reliable because of possible incomplete digestion and unsatisfactory resolution during chromatography.

It is interesting that the moles of 14C-pyruvate bound by borohydride reduction indicated three subunits. Kobashi, Lai, and Horecker (32) have reported that borohydride reduction has yielded low values for the number of binding sites and have dem- onstrated the presence of bound organic phosphate in preparations of rabbit muscle aldolase. Presumably, this bound phosphate occupies one of the active sites, which explains the low values obtained with borohydride reduction of the enzyme dihy- droxyacetone phosphate complex. The purity of W-dihydroxy-


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acetone phosphate has also been cited as a limitation in these experiments.

Four distinct sl)ecies were detected in the hybridization experiments with native and chemically modified KDPG aldolase. These results constitute strong evidence to support a three-subunit model because of the fact that the interpretation is independent of the parent molecular weight and the amount of protein used in an experiment. Interpretation of the hybridization experiments is influenced by the ability to separate the individual species. The following considerations make it unlikely that resolution of the hybrids was incomplete: (a) the large difference in charge between the native and chemically modified KDPG aldolase; (h) the resolving power of the gel method, and (c) the symmetry of the peaks. Furthermore, at the extremes of the gel column, enzymatic activity and radioactivity measurements provided additional sensitive indicators.

Maleic anhydride (33) was especially suited for the chemical modification of KDPG aldolase. Data were presented in the previous paper which showed that the protein does not dissociate after extensive maleylation (2). Twenty to 24 moles of maleic anhydride per mole of protein were incorporated, indicating complete reaction with the 21 lysine and N&-terminal residues. The fact that KDPG aldolase does not dissociate after maleylation suggests that the binding forces between subunits have not been disrupted. These results are in sharp contrast with those obtained with fructose-l, 6PZ aldolase where maleylation of 15% of the lysyl residues effected complete dissociation (34). The chemically modified KDPG aldolase was enzymatically inactive, yet the protein could be reconstituted after the dissociation and reassociation conditions described under “Experimental Pro- cedure.” The use of I%-maleic anhydride provided additional sensitivity in the detection of hybrid species.

Disc gel electrophoresis was used to substantiate the parent molecular weight obtained by physical methods reported in the previous paper (2). Hedrick and Smith (24) have noted that the technique is limited to proteins with a p1 of 6.0 to 6.5. KDPG aldolase has a p1 of 4.8 (2), and presented no problem; however, there are a limited number of standard proteins with a suitable pI in the molecular weight range of 60,000 to 150,000.

An exact value of the partial specific volume ($ is required to calculate the molecular weight of component polypeptide chains in dissociating media. The high density of dissociating media promotes large errors with small variation in V. Also, considera- ble disagreement exists as to the correction of fi) in comparing molecular weights in dilute salt solutions and dissociating media (35). Sodium dodecyl sulfate gel electrophoresis provided a method to determine the subunit molecular weight independent of the above limitations, as well as reflect heterogeneity with respect to size.

The history of fructose-l, 6-P, aldolase, which was thought to be the only example of a three-subunit enzyme until recently when it was shown to be composed of four identical subunits (29, 30, 36, 37), provokes some aversion to assigning a three-subunit model to KDPG aldolase. The experiments cited in this and the preceding paper were performed against this background. However, the data reported in these papers can only support a three-subunit model.

Ac&wwledgment-The authors wish to thank Mrs. Diana Ers- feld for her assistance in the gel electrophoresis experiments.

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19. YEMM, E. W., AND COCIUNG, E. C., Analyst, 80, 209 (1955). 20. SILVERSTEIN, E., AND BOYER, P. D., Anal. Biochem., 8, 470

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(1961). 22. DEAL. W. C., JR., Biochemistry, 8, 2795 (1969). 23. DAVIS, B. J., Ann. iV. Y. Acacl. Sci., 121, 404 (1964). 24. HEDIZICK, J. L., AND SMITH, A. J., Arch. Biochem. Bioph ys.,

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D. C. Robertson, R. H. Hammerstedt and W. A. WoodEVIDENCE FOR A THREE-SUBUNIT MOLECULE

Structure of 2-Keto-3-deoxy-6-phosphogluconate Aldolase: II. CHEMICAL

1971, 246:2075-2083.J. Biol. Chem.

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