Molecular properties of cis,cis-muconate cycloisomerase from Pseudomonas putida

J. ..VoZ.Bio2. (1974) 89, 651-662

Molecular Properties of cis,ci.s-Muconate Cycloisomerase from Pseudomonas putida GAD AJ?GAD~, SASHA EKGLARD~, BJDRS R. OLSEN*, CAKLOTA WOLBENSTEIK-TODEL’~ ASD I~OBI%RTWIGGISS* aDepartment of Biochemidy, Rutgels Medical School College of Nedicin.e und Dentidly of hTew Jersey, Piscafazcay, hr.J. 08854, U.S.A. bl)epartment of Biochemisty Albert Einstein College of Medici~ne Yeshiva University, Bronx, N. Y. 10461, U.S.A. (Received 21 Nay

1974)

cls&-Miiconatc cycloisomcrase (cia,cis-muconntc lactonizing enzyme, IaX 5..5.1.1.) was purifiod in crystalline form from %‘sezdomonas putida. Ultracentrifngation studies, ae well as gel filtration chromatography and clectrophoresis, indicate that the enzyme is an oligomcric protein of molecular lveight 2f&c)(JO (s~~,~ 12.20 x IO-l3 s), which is built of six homologous protorners of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in t.he electron rnicroscopc sueest that the cis,cis-muconatc cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (II:,). Each protorner has a diameter of 42.5 A and six protomen are associated in a structure with a trigonal antiprismatic geometry (a hcxorner D, octahedron). This modol could account for tho dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hcxameric enz-ymc molecule is surrounded by eight neighbouring enzyme molcculas, is described.

1. Introduction Muconatc

cpcloisomcrase

(also

called

f-is,&-muconate

la&onizing

enzyme,

or

4-

EC. 55.1 .I) catalyses the reversible converslon of cis,ci.s-muconaie to its lactone, y-carboxymethyl-Albutenolide. This reaction const,itutes one step in the sequence of the or& cleavage, /3-ketoxdipate enzymic pathway for the degradation of catechol (for recent reviews set Daglcy, 1971; Chapman, 1972: Stanier & Ornston, 1973). The enzyme was first described by Sistrom & Stan& (1954) and purified by Ornston (1966). Only limited information has been available on its molecular prop&ies and on the kinetics of the lactonization catalyzed by it, alt.hough some storeochcmical aspect,s of the reaction were described by Rvigad t Englard (1969). While the present paper was in preparation. a report has appeared (Meaghcr & Ornston, 1973) presenting additional informacarboxgmethyl!

tion

4-hydrosyisocrotonola.ct80ne

on the molecular

size of the suhunit,s

lyase

(c&y&zing),

of the enzyme.

We now report studies on the physicochemical properties of cis,cis-muconate cycloisomerase from Pseudomonas putida, particularly on its molecular weight and subunit t Prosent address: Rheumatic ;\‘a~ York, S.Y. 10016, U.S.A.

Diseases Study

Croup,

Sew York

University

Medical

Centor,

652

G. AVIGAD

structure electron

as determined microscopy. We

chemical

and kinetic

by gel-filtration, shall subsequently

aspects

of the reaction

ET

AL.

electrophoresis, publish detailed

ultracentrifugation studies

on the

and stereo-

catalyzed by this enzyme.

2. Materials and Methods (a) Growth of bacteria Pseudomonas putida A.3.12 (ATCC 12633) was obtained from Dr R. Y. Stanier. It was grown at 30°C in a sodium DL-mandelate medium as described by Sistrom & Stanier (1954). Small-scale growth in batches of 1 1 was conducted for 40 h in 2-1 Erlenmeyer flasks incubated on a New Brunswick Co. rotary shaker. The cells were collected by centrifugation for 15 min at 10,000 g, and then washed twice with 0.04 M-phosphate buffer (pH 6.8). Yield of packed cells was 7 to 8 g/l. Cells were kept at -20°C if not used immediately. Large-scale growth in batches of 120 1 was conducted in a Fermentation Design Inc. fermentor with constant stirring and forced aeration in the presence of Dow-Corning Q antifoam. A 3% starter volume of a 24-h culture was used for inoculation. Growth reached a maximum level after about 24 h (A,@’ = 3.0). The culture was rapidly cooled to 4°C and the cells collected by centrifugation in a Sharpless refrigerated supercentrifuge. Yield was 6 to 7 g packed cells per 1. Cells, stored in the deep-freezer at -20°C were later used for extraction of the enzyme without any additional washings. Frozen cells were usually used within 1 month of preparation, first being thawed at room temperature under a stream of tap water. (b) Materials Purified enzymes employed as standards in molecular size determination by gel filtration or electrophoresis were purchased from Worthington Biochemical Co. and Boehringer and Soehne GmbH, Mannheim. Ceruloplasmin was obtained from Dr A. Morell; bacterial cr-amylase from Dr E. J. Hehre ; X-malic dehydrogenase was a preparation described by Wolfenstein et al. (1969). Apoferritin and serum albumins were Pentex products, Miles Laboratories, Inc. cis,cis-Muconic acid was synthesized according to Elvidge et al. (1960). Bio-Gel agarose preparations were obtained from Bio-Rad Laboratories. Other chemical reagents used were obtained from commercial suppliers. (c) Ultracentrifugation

analysis

Sedimentation velocity ultracentrifugation was performed in a Beckman Spinco model E ultracentrifuge equipped with schlieren optics and using the AN-D rotor with standard 12 mm aluminum double-sector cells. Photographs were taken after the rotor attained a running speed of 52,870 revs/min at 2’C to 9’C in different experiments. Boundary positions were measured with a Nikon optical microcomparator. Diffusion coefficient was determined in the ultracentrifuge using a 12-mm double-sector cell with a capillary synthetic boundary centerpiece at a running speed of 4500 revs/min. Sedimentation equilibrium analyses using interference optics were carried out according to Yphantis (1964) using the same procedures described earlier for malate dehydrogenase (Wolfenstein et al., 1969). The partial specific volume (P) of the muconate cycloisomerase, was calculated from the amino acid composition (Cohn & Edsall, 1943) and found to be 0,735 ml/g. Calculations of sedimentation, frictional and diffusion coefficients, and values of molecular weight were made using standard equations as recommended by Sohachman (1959), Chervenka (1969) and Haschemeyer & Haschemeyer (1973). (d) Gel electrophoresis Disc-gel electrophoresis was performed according to Davis (1964) with a Canalco model 66 apparatus. Gels were prepared with a 7.5% aorylamide monomer concentration in Tris/glycine buffer (pH 8.8), and stained with 0.5% naphthol blue-black in 7% acetic acid.

CIS,CIS-MUCONATE

CYCLOISOMERASE

653

Estimation of molecular weight by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate was performed as recommended by Shapiro et al. (1967) and Weber & Osborn (1969). (e) Gel filtration Estimation of molecular weights of proteins was performed as recommended by Andrews (1965) and Fish et aE. (1969). The following columns were used: I. Bio-Gel A5M (1 cm x 50 cm) with a void volume (I’,) of 13 ml; II. Bio-Gel P300 (1 cm x 48 cm) with a 17s of 14 ml; III. Bio-Gel A5M (1 cm x 60 cm) with a 8, of 18 ml. Columns I and II were washed overnight with a 10 rmvr-Tris*HCl buffer (pH 7.2) and column III with 6 M-guanidine*HCl containing 1 m&r-mercaptoethanol (pH 5.0). Blue dextran 2000 (Pharmacia) and proteins in quantities of 2 to 3 mg were applied to columns I and II in 0.5 ml of 2% sucrose. Similar samples of proteins for column III were first dissolved in 0.5 ml of 6 Mguanidine*HCl and 0.1 M-mercaptoethanol at pH 8-O and allowed to incubate at 20°C for about 3 h before application to the column. Elution, at 4°C was facilitated at a rate of 3 ml/h with 10 mm-Tris*HCl buffer (pH 7.2) for columns I and II and with 5 m-guanidine*HCl (pH 50), for column III. Fractions of O-4 ml were collected and the protein was determined by measuring absorbance at 280 nm. (f) Amino acid analysis Samples of protein (3 to 5 mg) were hydrolyzed at 110°C in sealed, evacuated tubes for 20, 40 or 70 h (Moore & Stein, 1963). HCl was removed under reduced pressure and the hydrolysates analyzed in a Technicon amino acid analyser packed with Chromobeads C2 and using the standard gradient recommended in the Technicon AAA-1 manual. Halfcystine was determined after oxidation of the protein with performic acid (Hirs, 1967). Sulfhydryl groups in the absence or presence of 6 M-urea, were determined by spectrophotometric titration with p-chloromercury-benzoate (Swenson & Boyer, 1957) using the procedure described by Guha et aE. (1968). Alternatively, cysteine was also assayed colorimetrically using 5,5’-dithiobis-(2-nitrobenzoic acid) as described by Habeeb (1972). Tryptophan was estimated spectrophotometrically (Goodwin & Morton, 1946; Edelhoch, 1967). Protein was determined calorimetrically (Lowry &t al., 1951) using bovine serum albumin as a standard.

(g) Electron microscopy For the study of individual enzyme molecules, a 2% suspension of crystalline muconate cycloisomerase in 10 m-phosphate buffer (pH 7.0) and 30% saturated ammonium sulfate solution was diluted with 0.05 M-Tris.HCl buffer (pH 7.5) at 4°C to give a final protein concentration of 50 to 500 pg/ml. The enzyme was then examined in the electron microscope after negative staining as described by Olsen et al. (1973). Although several negative stains were employed in preliminary experiments, the best results were obtained with 1 y. potassium phosphotungstate (pH 7.5), or 1% uranyl acetate (pH 4.2). For electron microscopy of enzyme in crystalline form, the stock crystal suspension was diluted 5 times with 30% ammonium sulfate in 0.05 M-Tris.HCl buffer (pH 7.5). A drop of this diluted suspension was then applied to a copper grid previously coated with carbon, excess fluid blotted with filter paper, and the preparation was then stained negatively or shadow cast before examination in the electron microscope. Shadow casting was done with a JEMM vacuum evaporator. A preparation of 80% platinum/20% palladium was evaporated onto a grid with enzyme crystals at an angle of 70”. The grid was then coated with a thin film of carbon in a perpendicular direction. All specimens were examined in a JEMlOOB electron microscope. The magnification of the microscope was calibrated using the crystal spacing of liver catalase (Wrigley, 1968). Kodak electron image plates were exposed to the image. Plates with the recorded image of negatively stained crystals were analyzed in an optical diffractometer as described by Johansen (1972). The diffraotograms were recorded on Agfa 1F film, and the film developed with Kodak Dl 1 high-contrast developer. Light microscopy of the crystal suspension was performed with a Nikon MS inverted microscope using phase-contrast optics.

654

G. A’VIGAD

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AL.

For analysis of different views of individual enzyme molecules, the maximum and minimum dimensions of structures observed in electron micrographs were plotted in scatter diagrams and histograms as described by Wrigley et al. (1972) and Olsen et al. (1973). (h) Assay

of cis,cis-rnzLco?aate

cycloisomerase

nctivity

The standard spectrophotometric assay employed for scanning of enzyme activity (in 1.0 ml cuvettes with a 1.0 cm light path) was that described by Sistrom & Stanier (1954), and contained 0.1 mM-T&-neutralized cis,cis-muconate, 1 mar-MnCl, and 20 mnfTrisaHCl buffer as pH 8.0. However, since these conditions do not provide maximum rates of lactonization, the assay was modified as follows to enable the measurement of level. Reaction mixture (1.0 ml) in a 3-ml quartz cuvette with a l*O-cm rates at a B,,, light path, contained 20 m&r-Tris.HCl (pH S*O), 1.0 mM-neutralized &s&is-muconate and 2.5 mi?r-MnCl,. Quartz inserts (9 mm width) were employed to reduce the light path to 1 mm, and the decrease in absorbance at 260 nm was recorded in a Gilford model 2000 automatic recording spectrophotometer. A value for Asso of 1.7 X lo4 for c&,&s-muoonate acid (Sistrom & Starrier, 1954) was used to calculate the disappearance of the substrate due to its lactonization. One unit of enzyme activity was that amount of enzyme that catalyzed the lactonization of 1 pm01 cis,cis-muconatelmin at 25°C with the condition of assay described. (i) Purification

of cis,cismuconate

cycloisomerase

All procedures, unless otherwise stated, were conducted at 2°C. Wet, packed cells of P. putida (183 g) were dispersed in 900 ml of 0.01 M-phosphate buffer (pH 6-g), for 1 min in a Waring blender at high speed. After 10 min sonification in the Raytheon 10 kc sonic oscillator, cell debris was removed by centrifugation for 30 min at 15,000 g. The supernatant (850 ml) was retained; the precipitate wss dispersed in 800 ml of the same buffer for 2 min in a Waring blender, then centrifuged, and the supernatant combined wit,h the first one to provide the “crude extract” (1600 ml). Using a 10% (w/v) solution (591 ml), streptomycin sulfate in a final concentration of 2.68% was added with stirring to the crude extract. After an additional 30 min stirring on a magnetic plate, the material was centrifuged for 30 min at 18,000 g and the preeipit&e discarded. The streptomycin sulfate supernatant solution (2115 ml) then received 370 g solid ammonium sulfate to a final degree of saturation of 25%. After 1 h of stirring, the mixture was centrifuged for 30 min at 18,000 g and the precipitate removed. The ammonium sulfate was raised to 32% saturation by gradual addition of the solid salt (107 g). After 1 h, the precipitate was collected by centrifugation, as above. It contained most of the cycloisomerase activity; the supernatant could be retained for the purification of muconolactone d-isomerase (EC 5.3.3.4.) (0 rnston, 1966; Meagher & Ornston, 1973). The precipitate wss dissolved in 50 ml of 10 rnlvr-phosphate buffer (pH 6.8) and dialyzed for 48 h against two changes of 20 vol. of 5 mm-phosphate buffer (pH 6.8), containing 10 - 5 M-EDTA. The solution became slightly turbid and was clarified by centrifugation for 15 min at 15,000 g. The enzyme solution (90 ml) was applied to a Whatman DEAE-cellulose column (2.8 cm x 40 cm) previously washed with 5 mlvr-phosphate buffer (pH 6.8) containing lo-5 M-EDTA. The charged column then was washed with 200 ml of the same buffer. A linear gradient of salt concentration was applied to the column; this was prepared by use of a bottom flask thst contained 200 ml of the same buffer and a top flask that contained the same buffer but with 0.2 M-NaCl. With the aid of a peristaltic pump, fractions of 10 ml were collected at a rate of 50 ml/h. Microliter samples were taken from each tube to determine cycloisomerase activity. The enzyme usually eluted in fractions no. 15 to 45. The fractions containing most of the activity were pooled (280 ml) and concentrated under nitrogen pressure with the help of an Amicon Corp., model 202 stirred cell with a Die,flo ultrafilter UMlO or PMlO. To the concentrate (33 ml) a cold saturated solution of ammonium sulfate (15 ml) was added dropwise, with stirring, until incipient turbidity occurred (at about 337; saturation). The solution wss then stored at 2°C. Crystals started to appear within several

CIS,CIS-MUCONATE

655

CYCLOISOMERASE

hours as was indicated by the silvery sheen of the suspension when swirled. Additional saturated ammonium sulfate solution (2.6 ml) was added dropwise, and the suspension refrigerated overnight. For the purpose of recrystallization, the crystals were packed by 15 min centrifugation at 10,000 g and then dissolved in 12 ml of 10 m&r-Tris.HCl (pH 7.8). A saturated ammonium sulfate solution was added dropwise to incipient turbidity (at about 27% saturation of ammonium sulfate) and crystallization allowed to occur as described above. Subsequent recrystallizations could be carried out in the same manner. Crystals dissolved in 20 mMphosphate buffer (pH 7.4) could be recrystallized with similar results. A summary of the purification procedure is presented in Table 1.

TABLE

Purification

1

of muconate cycloisomerase Protein Volume (ml) hd ______

Fraction

1. Crude extract 2. Streptomycin sulfate supernatant 3. Dialyzed 25 to 32% saturation (NH&SOI 4. Concentrated DEAE-cellulose eluate 5. Crystals 6. First recrystallization 7. Second recrystallization

Units

Speoific activity?

Yield

1600 2115

13,150 11,080

36,470 35,870

2.7 3.2

100 98

90

2520

31,120

12.3

85

33 12 10 8

192 66 38 24

18,600 13,140 7400 4500

96.8 199.2 195.0 187.2

51 36 20 16

t When assayed at 0.1 miw-cis,cis-muconate and 0.66 mM-MnCl,, the values of spec. act. were approximately 50% of those recorded here, corresponding well with values reported by Ornston (1966) and Meagher & Ornston (1973).

3. Results (a) Purity

of the crystalline

enzyme

It is clear that the first crop of crystals has a maximum specific activity that does not change significantly by further recrystallization. In the light microscope the enzyme crystals were seen as thin plate-like structures with a square or rectangular appearance and orthogonal cleavage planes, similar to the observation of Ornston (1966). This appearance explained the finding (see below) that the crystals always were absorbed flat onto the carbon supports used for electron microscopy. When tested for catalytic activity, the enzyme isolated by this procedure showed a strong activaexperiments). This is in contrast to tion by Mn 2 + (Avigad Sr; Englard, unpublished the preparation described by Meagher & Ornston (1973) in which Mn2+ was present during the procedure of enzyme purification. The enzyme, dissolvedin 20 mM-Tris . HCl buffer (pH 8*0), and subjected to disc electrophoresis on polyacrylamide gels, moved as a single, clearly defined band. In the ultracentrifuge, the enzyme separated as a sharp homogeneous peak (Plate I). (b) Amino

acid composition

The amino acid composition of the enzyme (Table 2) was found in general to be in pattern to that described by Meagher & Ornston (1973) with some quantitative differences that are mostly minor. Notable is the present estimation of three or similar

656

G. AVIGAD

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TABLE 2 Amino

Amino acid

acid composition

20 h hydrolysis

of cis,cis-muconate

Residues per 1OOOt 40 h hydrolysis

cycloisomerase

70 h hydrolysis

Calculated residues per 42,000 M,

__Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenyblenine Tryptophan

34.6 20.0 66.6 84.1 48.0 47.0 121.2 42.1 96.4 122.5 9.2 61.7 17.5 62.1 118.2 17.2 25.2 7.08

35.6 24.9 65.4 79.2 46.6 42.8 118.5 43.1 87.2 122.1 8.2 68.6 14.7 67.9 120.3 17.9 26.7

40.0 22.7 66.4 83.0 45.2 42.3 113.5 44.3 91.8 122.3 8.6 69.9 16.4 69.9 118.6 17.9 26.3

14.0 8.6 26.7 31-9 18.3 17.9 45.9 16.7 35.0 47.1 3.53 26.5 6.2 26.6 45.9 6.6 10.1 2,7

t Each column represents everage value obtained from the analysis of 3 separate samples. $ Values of 3.5 to 4.0 half-cysteine residues were also found by analysis of cysteic acid contents efter oxidation of the enzyme by pcrformio acid prior to its acid hydrolysis. However, by spectrophotometric titration with p-chloromercurybenzoate or in the 5,5’-dithiobis(2nitrobenzoic acid) calorimetric reaction, only one cysteine residue per mole of enzyme monomeric unit could rapidly be titrated, whereas e second sulfhydryl w&s then titrated much more slowly. The same titration pattern of 2 sulfhydryl groups wes observed both for the native enzyme and in the presence of 8 M-urea. § Determined spectrophotometrically as indicated in Materiels and Methods.

four cysteine residues per 42,000 molecular weight unit of protein based on analysis of the acid hydrolysate, against the estimation of only two cysteine residues assayed by spectrophotometric titration procedures. In comparison, Meagher 87Ornston (1973), determined the presence of two cysteine residues for a monomeric unit of 40,000 molecular weight. (c) Ultracentr@hgaE

analyses

Sedimentation velocity studies of the enzyme have indicated an sZO,W value of 12.20 x lo-l3 s when extrapolated to infinite dilution of protein (Fig. 1). The diffusion coefficient calculated from the spreading of the boundary in an analysis of 2.47 mg protein/ml in 10 mM-phosphate buffer (pH 7.0) was calculated to be 4.35 x lo- ’ cm2 s- l. When these values were substituted in the Svedberg equation, the molecular weight of the enzyme was calculated to be 254,000. Sedimentation equilibrium studies of native enzyme (Fig. 2) indicated a calculated molecular weight of 248,500. When the sedimentation equilibrium analysis was carried out in the presence of 6 M-guanidine*HCI, it was found to dissociate into a homogeneous molecular species of 41,600 molecular weight (Fig. 2).

PLATI 1. Sedimentation of muconate cycloisomerase in the ultracentrifuge. tained 3.7 mg protein/ml of 10 mM-phosphate buffer (pH 7.0). Pict,ures shown 30 min aft,er the r&or speed reached a constant 52,870 revs/min at 9°C.

The aample conwere taken 15 and

PLATE II. Electron micrograph showing shadow casting; magnification x 137,500; kngstate, magnification x 275.000. Inset symmetry.

crystals of cis,cis-muconate cycloisomerase; (a) after (b) after negative staining with potassium phosphois the optical diffraction pattern of (b) showing 4.fold

PLATE III. cis,cis-Muconate cycloisomerase negatively stained with potassium phosphotungstate. (a) Edge of enzyme crystal; magnification x 165,000. (b) Single layer of enzyme molecules formed from dissolving crystals on the grid during the procedure of negative staining; magnification x 275.000.

PLATE IV. cis,cis-Muconate cycloisomerase dissolved in 0.05 ;\r-Tris.HCl buffer negatively stained with polzwsium phosphotungstate. (a) Magnification x 110,000. tion x 440,000.

(pH 7.5) amI (b) Magnifioa-

PLATE V. Different, views of a model proposed for cis,cis-muconate cycloisomerase. The enzyme molecule consists of six sphericel protomers each with a diameter of 42.5 .& forming n trigonal antiprism (or a hexamer in a D, octahedron). (a) Molecule viewed along an axis about 45” from the 3.foid axis and 30” from a 2.fold axis. (b) Molecule viewed along the 3.fold axis. (c) iMolrculo viewed along a 2.fold axis.

Top view

Side view

------

/

/

/

/

/’

\

t ‘\,85fi

\\

Cb)

-----\

\

\

\

‘Cl

\

\

/+ \

103 II

\ “\/.

~‘LATE VI. (a) A crystal of cis,cis-muconate cycloisomerase negatively stained with potassium phosphotungstate (magnification x l,lOO,OOO) compared with a pattern obtained by a picture of a model of the crystal built with trigonal antiprismatic molecules as shown in Plate V. (b) Schematic representation of the arrangement of the hexameric enzyme molecules in the crystal structure seen in (a). Except in the case of crystal surfaces, each enzyme molecule within the crystal has contact with eight neighboring enzyme molecules.

CIS,CIS-MUCONATE

FIG. 1. Sedimentrttion coefficient at different enzyme concentration

657

CYCLOISOMERASE

of muconate cyoloisomerase. were carried out as described

Sedimentation velocity in Materials and Methods.

studies

(d) Estimation of molecular weight by gel electrophoresis and gel jiltration Electrophoresis of the muconate cycloisomerase in the presence of sodium dodecyl sulfate indicated the presence of one species of protein with a molecular weight of about 44,000 (Fig. 3). Gel filtration of the native enzyme on two columns (Fig. 4) indicated a molecular weight of 250,000 to 260,000. In the presence of guanidine.HCl, the enzyme dissociated into a molecular species of about 42,000 as determined by gel filtration (Fig. 4). of cis,cis-muconate cycloisomerase crystals After shadow casting of crystals with platinum/palladium all the crystals observed in the microscope showed a pattern of parallel white lines (Plate 11(a)) spaced 117f3 A apart. Since the structure seen after shadow casting is due to deposition of the heavy metal on top of the crystal, we may conclude that the top surface of the crystal has a periodic structure with a regular spacing of about 120 A. Negative staining of the crystals with potassium phosphotungstate demonstrated a periodic pattern (Plate II(b)) with 4-fold symmetry. As indicated in Plates II(b) and (e) Electron microscopy

464

46-6

468

(a)

470

472

458

r?CRll*

462

466

470

(0)

FIG. 2. Molecular weight determination of cis,cis-muconate cycloisomerase by sedimentation equilibrium. (a) Native enzyme (0.5 mg/ml) in 0.1 ix-Tris.HCl (pH 7*4), ZO”C, with a rotor speed of 14,280 revs/min. Similar results were obtained also in 0.1 M-phosphate buffer (pH 7.0). (b) The enzyme (0.6 mg/ml) in 6 M-guanidine.HCl, 0.1 x-p-mercaptoethanol (pH 7.0), with a rotor speed of 39,400 revsjmin.

-I-_--L

20

40

60 rielotlve nlyJtlor1

80

100

FIG. 3. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Semilog plot of molecular weight against distance of migration relative to hemoglobin. Reference proteins: a, bovine serum albumin dimer; b, ovalbumin dimer; o, bovine serum albumin; d, ovalbumin; e, bovine heart S-malio dehydrogenase; f, oarboxypeptidase A; g, trypsin; h, hemoglobin; M-LAC, cis,cismuoonste oyoloisomerase.

IO !b2 14

I6

18 20 22 24 ve / i/o

26

28

FIG. 4. Determmetion of molecular weight of muoonate oyoloisomerase by gel filtration. Analytical procedure was as described under Materials and Methods. The abscissa represents the ratio of V’e (elution volume) to V0 (void volume), and the ordinate log molecular weight. Curve A, pattern of elution from column I, Bio-Gel ABM; curve B, pattern of elution from column II, Bio-Gel P300; curve C, pattern of elution from column III, Bio-Gel A5M in presence of guenidine.HCI. Reference molecules: 1, blue dextran; 2, beef liver glutamio dehydrogenese; 3, Escherichia 00% /3-galaotosidase; 4, jack bean urease; 5, horse spleen rtpoferritin; 6, yeast phosphofructokinase; 7, beef liver oatalase; 8, rabbit muscle pyruvate kinase; 9, rabbit muscle aldolase; 10, horse liver alcohol dehydrogenase; 11, beef heart S-malio dehydrogenase; 12, bovine serum albumin; 13, 15, milk xanthine oxidase; Bacillus sub&e var. amylosaceharitic~ a-amylase ; 14, ovalbumin; 16, human serum ceruloplasmin; 17, pig kidney D-amino acid oxidase; 18, human serum albumin; 19, a-chymotrypsinogen A; 20, egg lysozyme; 21, oytoohrome c; M-LAG, cis,cis-muoonate cyoloisomerase.

GIS,CIS-MUCONATE

659

CYCLOISOMERASE

III(a) the observed two-dimensional square lattice may be described by the periodic dimensions of 120*4-&1*0 A and 84.8f1.2 A disposed at an angle of 45”. Lines of half-spacing values produced a pseudo-repeat distance of 60 A. The optical diffraction patterns of the crystals clearly shows the 4-fold symmetry (Plate II (inset)). Of special interest is the observation that in certain areas where crystals were dissolving, single layers of enzyme molecules seemed to be arranged in a square pattern with a periodicity of about 110 A (Plate III(b)). (f) Electron microscopy of single molecules Electron microscopy of individual enzyme molecules showed rounded particles with a diameter of about 90 A (Plate IV(a)). At higher magnification (Plate IV(b)), a number of different shapes could be observed that can be interpreted to represent views of different projections of the molecules being adsorbed onto the carbon support in different orientations. Some of the views were rectangular, some particles showed a pentagonal or hexagonal structure, and some molecules showed a double-bar appearance. In order to approach the question of molecular substructure in a more systematic way, we measured the smaller and larger dimensions, respectively, in two perpendicular directions of all molecules in randomly selected fields in different micrographs. The measurements were made on micrographs at, a magnification of 275,000 and the data obtained were plotted in a scatter-diagram. As can be seen from Figure 5, the molecular dimensions fell into distinct groups and the three most frequent views of the molecules had the approximate dimensions of 110~ 110 A, 110x90 A, and 110 x 75 A.

.‘“. y.i;;;, ..-..,$..A. .: .~%~~“;:: : (,. : ; .. . . +;.<.; .. ..)$ p . .,. . .. ..

.:

I

70

I

SO

I

I

I

I

0

:>.:: .:y.:

,. ..

,yfj

...

. ... ;:;.y: A..

:

I

90 100 110 Largest dimension ( 1 )

. ... ,,_

.

.:

.

. :.

<.

I

.

.:.

I

120

I

I

130

FIG. 5. Scatter diagram showing the diskibution of dimensions measured in electron micrographs of negatively stained cis,cis-muconate cycloisomerase molecules. Diagonal line indicates structures v&h an axial ratio of 1.

660

G. AVIGAD

ET

AL,

4. Discussion Molecular weight determinations of cis,cis-muconate cycloisomerase using several methods (Table 3) indicate that it is an oligomeric globular protein with a molecular weight of about 252,000, which dissociates in sodium dodecyl sulfate or guanidine .HCl solutions into homogeneous polypeptide protomers of about 42,000 molecular weight. The most obvious assumption therefore will be that the native, active form of enzyme is a hexamer built of six identical subunits (protComers). Previous findings based on gel chromatography suggested that the enzyme, with a molecular weight of about 220,000, could dissociate into an undetermined number of subunits of about 40,000 molecular weight (Ornston, 1966; Meagher & Ornston, 1973). The hexameric subunit structure of the enzyme based on physico-chemical evidence, finds support in the electron microscopic observations made in the present study. Since it is expected that oligomeric enzymes containing chemically equivalent subunits are symmetric or forming “closed” structures (Klug, 1967), the six subunits of the cycloisomerase would therefore be expected to be arranged according to the requirements of point-group symmetry (Matthews & Bernhard, 1973). Two types of structures are possible. (a) The subunits are arranged in a ring with a single 6-fold rotation axis (cyclic point group symmetry 6 (C,)), or (b) they form two antiparallel triangles. The latter type of structure belongs to dihedral point group 32 (OS) symmetry group. Our micrographs of single molecules and their observed dimensions do not support the theory of a ring structure with a g-fold rotation axis. The pictures are, however, fully compatible with a structure having a trigonal antiprismatic geometry, or of a hexamer D, octahedron for the enzyme (Klotz et al., 1970; Haschemeyer & Haschemeyer, 1973). In such a case, the molecule would have one 3-fold symmetry TABLE 3 Physical

properties

of cis,cis-mT:conate cycloisomerase

Property Native enzyme Sedimentation coefficient (so,,,,) Diffusion coefficient (DzO,w) Frictional coefficient (f/fo)t Stokes’ radius3 Average diameter, electron microscopy§ Partial specific volume ( V) 11 Molecular weight, Sedimentation velocity Sedimentation equilibrium Gel filtration Subunits, molecular weight Sedimentation equilibrium in guanidine . HCI Gel filtration in guanidine . HCl Gel electrophoresis in sodium dodecyl sulfate

Value

12.20x lo-= s 4.35 x lo-? omz 8-l 1.152 48.4 A 95 a 0.735 ml g-1 254,400 248,500 260,000 41,600 42,000 44,000

Experimental values are those obtained in the present study. Calculations were made according to standard equations (cf. Schaohman, 1959; Haschemeyer & Haschemeyer, 1973). T Calculated for a molecular weight of 252,000. $ Calculated from the diffusion coefficient (of. Siegel & Monty, 1966). 5 Measured after negative staining. jj Calculated from the amino acid composition.

CIS,CIS-MUCONATE

661

CYCLOISOMERASE

axis and three identical 2-fold axes with the latter all lying in a plane perpendicular to the S-fold axis. The double-bar appearances would represent views along axes lying in a plane parallel to the planes of the two triangles (Plate V). If the molecule is viewed down the 3-fold axis, one would expect to see hexagonal structures, or if the negative-stain film is thin, triangles. It is interesting to note that the observed average diameter of the enzyme corresponds well with the estimated Stokes’ radius of the protein determined on the basis of physical determinations (Table 3). Further evidence, which is consistent with the proposed model for the tertiary structure of muconate cycloisomerase, can be derived from the analysis of crystalline single sheets of enzyme molecules, as well as of thicker crystals. A ring-shaped molecule with six subunits is difficult to reconcile with the h-fold symmetry observed in the crystals. However, a trigonal antiprism where the average diameter of each subunit is about 55 to 60 A would easily produce the observed line patterns and periodicities by being arranged as indicated in Plate VI. This would lead to a regular arrangement of subunits protruding from the top surface of the crystal with a periodicity of about 120 A, in full agreement with the results obtained by shadow casting. It should be noted that in constructing a model for single enzyme molecules, we have used primarily dimensions obtained by measuring spacings and periodicities observed in negatively stained crystals. In view of the difficulty of accurately defining the edges of negatively stained single enzyme molecules that were in solution, measurement of crystals obviously provides the more accurate estimate of molecular dimensions by yielding well-defined spacings. The molecular dimensions obtained for the proposed model in effect agree well with the approximate values for the minimum and maximum diameters measured in micrographs of single, negatively stained cis,cis-muconate cycloisomerase molecules in solution. The molecular model proposed here is in several aspects similar to the models proposed for glutamate dehydrogenase (Josephs, 1971; Josephs et al., 1972) and the proteins edestin and excelsin from hemp seed and brazil nuts, respectively (Schepman et al., 1972). One of us (R. W.) was supported by the College of Medicine and Dentistry of New Jersey, Rutgers Medical School Pre-Doctoral Summer Fellowship Program. This investigation

was supported

in part

by grant

GB6053

from

the National

Science

Foundation.

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