JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 690-697Copyright © 1977 American Society for Microbiology

Vol. 129, No. 2Printed in U.S.A.

Purification and Properties of Homoprotocatechuate 2,3-Dioxygenase from Bacillus stearothermophilus'

MOIDEEN P. JAMALUDDIN2Department ofBiochemistry, College ofBiological Sciences, University of Minnesota, St. Paul,

Minnesota 55108

Received for publication 16 August 1976

The enzyme 3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decycliz-ing) (homoprotocatechuate 2,3-dioxygenase) was purified from the thermophilicorganism Bacillus stearothermophilus, grown with 4-hydroxyphenylacetic acidas a source of carbon. The enzyme appeared to be homogeneous as judged bydisc-gel electrophoresis and sedimentation equilibrium measurements. The av-erage molecular weight determined by three independent procedures was106,000; the protein was globular and was dissociated in sodium dodecyl sulfateto give a species of molecular weight 33,000 to 35,000. The enzyme was fairlystable on heating and showed maximal activity at about 57°C. An Arrhenius plotofKm for homoprotocatechuate was concave upward, with a break at 32°C; anincrease in AHIt above this temperature was compensated by lower values of-ASt. Several properties of this enzyme are contrasted with those reported forhomoprotocatechuate 2,3-dioxygenase purified by other workers from Pseudo-monas ovalis.

Homoprotocatechuate 2,3-dioxygenase (3,4-dihydroxyphenylacetate: oxygen 2, 3 - oxidore-ductase [decyclizing]) catalyzes fission of thebenzene nucleus of homoprotocatechuate togive 5-carboxymethyl -2 -hydroxy-cis, cis -mu-conic semialdehyde (Fig. 1). This enzyme, crys-tallized from Pseudomonas ovalis (9, 17), wasdescribed previously (15), and has been as-signed the number EC 1.13.11.15. Evidence forfission between C2 and C3 of the benzene nu-cleus was presented by Adachi et al. (1) andBlakley et al. (2) and is in accordance with themetabolic studies of Sparnins et al. (18). It maybe mentioned that the Committee on Biochemi-cal Nomenclature (3) has also assigned thenumber EC 1.13.11.7 to a homoprotocatechuateoxygenase that is shown as catalyzing fissionbetween C3 and C4. However, the sole refer-ence given is to Kita et al. (10), who studiedhomoprotocatechuate 2,3-dioxygenase (EC1.13.11.15), and it therefore appears that theentry EC 1.13.11.7 is redundant. The presentpaper describes the purification of homoproto-catechuate 2,3-dioxygenase (HP dioxygenase)from the thermophilic microorganism Bacillusstearothermophilus. The properties of the en-

' Address reprint requests to: Stanley Dagley, Depart-ment of Biochemistry, University of Minnesota, St. Paul,MN 55108.

2 Present address: Department of Microbiology and CellBiology, Indian Institute of Science, Bangalore 560012, In-dia.

zyme from this source were found to differ inseveral respects from those reported for the en-zyme isolated from Pseudomonas.

MATERIALS AND METHODSIsolation of the organism and conditions of cul-

ture. The bacillus was isolated by Mark Elstad fromgrass cuttings fermenting at 65°C. Pasteurization at80°C for 10 min was followed by selective enrichmentfor growth with phenylacetic acid at 65°C. The orga-nism was identified as B. stearothermophilus (7).Stock cultures were maintained on nutrient agar(Difco) slants, stored at 4°C, and subcultured atintervals of 2 weeks. The growth medium, adjustedto pH 7 with NaOH, contained (per liter):K2HPO4 * 3H20, 4.25 g; NaH2PO4 H20, 1.0 g; NH4Cl,2.0 g; MgSO4- 7H20, 0.2 g; FeSO4 7H20, 0.12 g;MnSO4 H20, 0.03 g; ZnSO4 7H2O, 0.003 g; CoSO4,0.001 g; vitamin-free Casamino Acids (Difco), 0.05 g;yeast extract (Difco), 0.05 g; and 4-hydroxphenyla-cetic acid, 0.5 g. A stationary culture (50 ml) wasgrown at 65°C and used to inoculate 1 liter of me-dium at 60°C in a shaken 2-liter Erlenmeyer flask,

HOOC CH2

OH

OH

H OOC - CH2

02 1'FCHO

L<<COOH

OH

FIG. 1. Reaction catalyzed by HP dioxygenase.690

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HOMOPROTOCATECHUATE DIOXYGENASE 691

and this culture was used as inoculum for 16 liters ofmedium in a carboy provided with forced aeration at60°C. When the absorbancy of the culture reached0.45, an additional 8 g of4-hydroxyphenylacetic acidwas added and incubation continued. Cells wereharvested by centrifugation at an absorbancy of 0.8and washed once with 0.05 M K+-Na+ phosphatebuffer (pH 7.0) containing 1 mM ethylenediamine-tetraacetic acid (EDTA), 0.1 mM dithiothreitol, and0.2 g of sodium azide per liter. Cells were keptfrozen at -20°C until needed. The yield of cellsfrom 16 liters of culture was 10 to 13 g (wet weight).

Materials. 3,4-Dihydroxyphenylpropionic acidwas a gift from Peter Chapman. All other substratesand coenzymes were from sources specified by Spar-nins et al. (18). Glyceraldehyde phosphate dehydro-genase (rabbit muscle) was from Worthington Bio-chemicals Co.; other standard proteins for gel filtra-tion chromatography and electrophoresis were fromSigma Chemical Co., which was also the source ofhydroxylapatite. Diethylaminoethyl (DEAE)-cellu-lose-Sephadex A-50 was from Pharmacia FineChemicals, Inc.Enzyme assay and kinetic parameters. HP dioxy-

genase activities were assayed at 21°C essentially asdescribed by Kita (9) by following the increase ofabsorbancy at 380 nm (A3M) due to the appearance ofring-fission products. Measurements were madewith a Gilford-modified Beckman spectrophotome-ter equipped with thermostable cell jackets and au-tomatic cuvette exchanger. Mixtures for the assaycontained, in a total volume of 1 ml, 49 ,umol of 0.05M K+-Na+PO4 buffer (pH 7.5), 0.1 ,umol of 3,4-dihy-droxyphenylacetate (homoprotocatechuate), and asuitable addition ofenzymoe One unit ofenzyme wasdefined as the amount that catalyzed the formationof 1 ,umol of ring-fission product per min under theconditions specified. In kinetic experiments not in-volving variations of substrate concentration, 98,umol of phosphate buffer (pH 8.2) was used with 1,umol of homoprotocatechuate. Temperature varia-tions, when required, were achieved by circulatingwater from a constant-temperature bath (+ 0.5°C)through the spectrophotometer cell jackets. Appar-entKm and Vma. values were calculated from initialreaction rates, measured for at least four concentra-tions in the range. 2 to- 10 Km for the carbon sub-strates used. Accurate detetminations of velocitiescould not be made at lower concentrations. All Line-weaver-Burk plots were linear; apparent K. andVma. values, with their standard errors, were esti-mated by fitting initial velocities to the Michaelis-Menten equation according to the procedure ofWilkinson (21).

Disc-gel electrophoresis. For disc-gel electropho-resis, the method of Davis (4) was used, with 7.5%polyacrylamide gels at pH 8.3 and at room tempera-ture. Gels were stained for protein by immersion inCoomassie brilliant blue R (Sigma) for 1 h andwere destained with a gel destainer (Canalco) in 7%acetic acid. Enzyme activity was located by placinggels in 5 ml of 0.05 M phosphate buffer (pH 7.5)containing 10 ,umol of homoprotocatechuic acid. Theposition of the enzyme was shown within 3 min bythe development of a yellow band, which subse-

quently dissipated slowly. Electrophoresis in thepresence of sodium dodecyl sulfate (SDS) was per-formed as described by Weber and Osborn (20). Re-duced and nonreduced samples of enzyme pro-tein were prepared as follows. The sample (1 ml)of dialyzed enzyme (1 mg of protein) in 0.0125M tris(hydroxymethyl)aminomethane (Tris)-hydro-chloride buffer (pH 7.5) containing 1% SDS wasdivided into two portions, to one ofwhich was added(3-mercaptoethanol to give a final concentration of10%. Both samples were incubated at 99°C for 25min, at which time 10 to 15 ,ul of 0.05% bromophenolblue and one drop of glycerol were added to each;about 30 ,ug of sample was applied to the gel.

Analytical ultracentrifugation. A Spinco model Eanalytical ultracentrifuge was used with an AnDrotor. Sedimentation velocity in a 12-mm double-sector cell (20°C; 56,000 rpm) was measured by usingschlieren optics as described by Schachman (16).The cell contained 0.7 mg of protein in 1 ml of Tris-hydrochloride buffer (pH 7.5) to which was added 0.1M NaCl, 1 mM ethylenediaminetetraacetic acid, 0.1mM dithiothreitol, and 0.02% sodium azide. Themeniscus depletion method of Yphantis (22) wasused in sedimentation equilibrium experiments.The protein (0.33 mg/ml) was dissolved in the samebuffer as that used for measurements of sedimenta-tion velocities, except that 0.17 M NaCl was present.For these experiments, a 12-mm double-sector cellwas equipped with interference window holders andsapphire windows. At equilibrium the Raleighinterferograph was taken, and measurements ofvertical fringe displacements were made with aGaertner comparator for a series ofradial distances.Amino acid analysis. The amino acid composition

of the purified protein was determined before andafter oxidation with performic acid (13). Hydrolysiswas carried out in 6 N HCI at 110°C for 22 h inevacuated, sealed Pyrex tubes. After removal ofHCI, the sample was analyzed by the procedure ofMoore and Stein (14). Tryptophan was not deter-mined.

Determination of thermodynamic parameters.Calculations of enthalpy of activation (AHt) andentropy of activation (St) were based upon theequation for the rate constant:

kT ASt/R e iHtIRTkr= kT-e s*Re AR

It follows from this equation that the plot of log (krlT) against 1T is linear; from the slope, AHt can becalculated, and the intercept (1T = 0) gives AStwhen Boltzman's constant (h) and Planck's constant(k) are assigned their numerical values. Experi-mental values of kr (= V^I/[E]) were calculated inunits of second-' by expressing enzyme concentra-tion ([El) as millimolar and Vm,,, as millimoles persecond (the molecular weight of the enzyme isshown later to be 1.06 x 105). Free energy of activa-tion (AGt) can be obtained from the relationshipAGt = AHt - TASt. If it is assumed that the recip-rocal of the Michaelis constant for homoprotocate-chuate (11Km) measures an equilibrium constant ofbinding, the following relationships and procedures

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692 JAMALUDDIN

may be used: free energy of binding, from AG = RTln Km; enthalpy of binding (AH), from a plot of ln K,..against 1T by taking the slope of a tangent to thecurve at the temperature specified; and entropy ofbinding (AS), from AS = (AH - AG)IT.

Purification of HP dioxygenase. Frozen cells (25g) were thawed and suspended in 100 ml of 0.05 MK+-Na+PO4 buffer (pH 7.0) containing 1 mM EDTA,0.1 mM dithiothreitol, and sodium azide (0.2 g/liter).This solution will be referred to as "supplementedphosphate buffer (pH 7.0)." Cells were broken byexposure for 15 min to the output of a BransonSonifier as described previously (18). Crude ex-tracts, containing 19 to 23 mg of protein per ml, were

obtained by centrifugation at 12,000 x g for 30 min,treated with protamine sulfate, and then fraction-ated with ammonium sulfate.A solution of protamine sulfate (20 g/liter) was

added to crude extract, with magnetic stirring, inthe ratio of 10 ml of solution to 1 g of extract protein.The mixture was stirred at 4°C for 30 min and theprecipitate was removed by centrifuging. To theclear yellow supernatant was added solid(NH4)2SO4, with stirring, to 0.65 saturation. Afterstanding overnight, the precipitate was collected bycentrifugation, dissolved in 10 ml of supplementedphosphate buffer (pH 7.0), and dialyzed for 3 h atroom temperature with three changes (each 250 ml)of the same buffer. The dialyzed enzyme was thenchromatographed on DEAE-cellulose-Sephadex A-50, first in supplemented phosphate buffer (pH 7.0)and then in Tris-hydrochloride buffer (pH 7.5), as

described below.The enzyme was applied to a column of DEAE-

Sephadex A-50 (2.2 by 28 cm) equilibrated with sup-plemented phosphate buffer (pH 7.0). The columnwas washed with 100 ml of buffer and eluted with a

linear gradient of 0 to 0.5 M NaCl in a total of 500 mlof buffer. The flow rate was 30 ml/h and 5-ml frac-tions were collected. The enzyme was eluted be-tween 0.35 and 0.45 M NaCl. Fractions having spe-cific activities of 0.13 or more were mixed together,cooled to 4°C, and brought to 2 mM with respect toEDTA, and the protein was precipitated by the addi-tion of (NH4)2SO4 to 0.65 saturation. After stirringfor 3 h, the precipitate was collected by centrifuga-tion and dissolved in 3 ml of 0.0125 M Tris-hydro-chloride buffer (pH 7.5) containing 1 mM EDTA, 0.1mM dithiothreitol, and sodium azide (0.2 g/liter).The solution was dialyzed against 200 ml ofthe same

buffer for 1 h and then overnight with a change ofbuffer.

This enzyme solution was then applied to a col-umn of DEAE-cellulose-Sephadex A-50 (2.2 by 44cm) that had equilibrated with the buffer used fordialysis in the previous step. The column was

washed with 200 ml of this buffer containing 0.2 MNaCl, and a linear gradient of 0.2 to 0.5 M NaCl in500 ml of Tris-hydrochloride buffer (pH 7.5) was

then applied. The enzyme was eluted between 0.3and 0.4 M NaCl. Fractions having specific activitiesof 0.24 and greater were mixed together, cooled to4°C, brought to 2.0 mM with respect to EDTA, andprecipitated with (NH4)2S04. The precipitate was

collected by centrifugation, dissolved in 2 ml of 0.02M K+-Na+PO4 buffer (pH 7.0) containing 1 mMEDTA and 0.1 mM dithiothreitol, and then dialyzedagainst the same buffer for 1.5 h with three changes(50 ml each) of buffer.The enzyme at this stage contained traces of con-

taminating impurities as judged by disc-gel electro-phoresis, and to obtain preparations that showed a

single band with coincident enzymic activity theprotein solution was submitted to chromatographyon hydroxylapatite. However, this step also resultedin a loss of enzyme with no increase in specificactivity (Table 1). This observation and the rela-tively small increases in specific activities observedthroughout suggest that each step in purificationwas compensated to some extent by an irreversibledecrease in enzymic activity. Unlike HP dioxygen-ase from P. ovalis (9), activity could not be regainedby incubation with Fe21 ions. The procedure forchromatography on hydroxylapatite was as follows.The dialyzed enzyme from the previous step was

applied to a column of hydroxylapatite (2.2 by 11 cm)equilibrated with the dialysis buffer. The columnwas washed with 70 ml of the starting buffer fol-lowed by 70 ml of 0.1 M sodium phosphate buffercontaining 1 mM EDTA and 0.1 mM dithiothreitol.A linear gradient of 0.1 M to 0.4 M sodium phos-phate (pH 7.0) was then applied in a total volume of260 ml. Fractions (3 ml) were collected, and theenzyme was eluted between 0.15 and 0.2 M sodiumphosphate. Fractions having specific activities be-tween 0.175 and 0.27 were mixed together and di-alyzed against 0.02 M Tris-hydrochloride buffer. Theenzyme at this stage of purification appeared to behomogeneous as judged by disc-gel electrophoresisand analytical ultracentrifugation.

TABLE 1. Purification ofHP dioxygenase

Protein Total ac- Sp act Purifica- YieldFraction (mg) ~~~~tivity (Um) tionFraction (mg) ~~~~~~~(U)(Um) (fold)

Crude extract .......................... 2,500 78 0.031 (1) (100)Protamine sulfate treatment ...... ...... 1,360 70 0.052 1.6 90Ammonium sulfate precipitation ........ 800 50 0.063 2.0 64DEAE-cellulose-Sephadex chromatogra-phy (pH 7.0) ......................... 157 25.3 0.161 5.2 32

DEAE-cellulose-Sephadex chromatogra-phy (pH 7.5) ......................... 39.8 10.9 0.275 8.9 14

Hydroxylapatite column chromatography 6.0 1.5 0.250 1.9

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HOMOPROTOCATECHUATE DIOXYGENASE 693

RESULTSMolecular properties of HP dioxygenase.

Values for the molecular weight were calcu-lated from separate experiments by using sed-imentation velocity, sedimentation equilib-rium, and Sephadex chromatography data.Sedimentation velocity measurements gaves20.W = 7.05 x 1013 s andDOw = 5.8 x 10-cm2/s. From the amino acid composition (seeTable 2), the partial specific volume (0) wascalculated to be 0.723 cm3/g; tryptophan (v =

0.74) was not determined. From these values,a molecular weight of 108,000 was calculatedby the Svedberg equation. In sedimentationequilibrium experiments, a plot of log (fringedisplacement) was linear over the wholelength of the cell (Fig. 2), confirming the ho-mogeneity of the protein species. After a lin-ear least-squares analysis ofthe data, a molec-ular weight of 101,000 was obtained.

In a Sephadex G-200 column calibrated withglyceraldehyde phosphate dehydrogenase, bo-vine albumin, ovalbumin, and chymotrypsinA, the enzyme eluted in the position expectedfor a globular protein of molecular weight110,000. An average molecular weight of106,000 was calculated from the values ob-tained in the three methods of molecularweight determination.From the values ofv and s 0,W and the molec-

ular weight of 108,000 (sedimentation veloc-ity), the frictional ratio fifo = 1.14 was ob-tained. This value approximates that expectedfor an unsolvated sphere. Disc-gel electropho-resis of the reduced, or unreduced, enzymeprotein in presence of SDS revealed the pres-ence of one and the same major protein band.The electrophoretic mobility of this proteinband corresponded to a molecular weight of34,000 + 1,000 when compared with bands ofreduced, SDS-treated bovine albumin, ovalbu-min, and chymotrypsinogen A, having molec-ular weights of 67,000, 45,000 and 25,000, re-spectively. This result suggests that HP dioxy-genase ofB. stearothermophilus is composed ofidentical subunits of molecular weight be-tween 33,000 and 35,000. The observation thatunreduced SDS-treated enzyme also showedthe same electrophoretic mobility indicatesthe absence of disulfide bridges between sub-units. The amino acid analysis showed thatmethionine and half-cystine were present inthe smallest ratios. A minimal molecularweight of 8,126 was calculated for one me-thionyl residue, without accounting for trypto-phanyl residues. Assuming the presence of 12methionyl residues per molecule, the aminoacid composition is given in Table 2.

KO+

zw

LU

u 2.5

a.

w

z

CL0

-it05.5

r2 (cm2)51.0

FIG. 2. Sedimentation equilibrium data of puri-fied HP dioxygenase. Experimental details are givenin the text.

TABLE 2. Amino acid composition ofHPdioxygenasea

Amino Residues/ Amino Residues/acid molecule acid molecule

Half-cystineb 11 Methionine 12Aspartic acid 91 Isoleucine 39Threonine 35 Leucine 79Serine 43 Tyrosine 35Glutamic acid 113 Phenylala- 46Proline 39 nineGlycine 67 Histidine 34Alanine 81 Lysine 48Valine 67 Arginine 53

a Number of residues per molecule was calculated on thebasis of 12 methionyl residues.

b Determined as cysteic acid after performic acid oxida-tion.

The ultraviolet spectrum of purified HPdioxygenase showed Xmax at 275 nm and Xmin at247.5 nm in 0.0125 M Tris-hydrochloride (pH7.5). The ratio A280/A260 was 1.53.Enzymatic properties. The enzyme was

quite heat stable. In 0.02 M Tris-hydrochloride(pH 7.5) from which air had been displacedwith N2, the enzyme (1 mg/ml) lost 5% of itsactivity in 2 h at 630C. The half-life of theenzyme at 880C was 10 min. A shallow pH-activity profile, with an optimum between pH8.4 and 8.7, was obtained for the enzyme inTris-hydrochloride at 29°C with 1.0 mM homo-protocatechuate; 78% of the optimal activitywas shown in this buffer at pH 7.09 and also atpH 9.3 in glycine-sodium hydroxide buffer. Al-though retaining activity during the heattreatment described, the enzyme lost activitywhen stored. Of various reagents used to pro-tect against loss, including 10% glycerol, the

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most successful was 0.0125 M Tris-hydrochlo-ride buffer (pH 7.5) containing 1 mM EDTA,0.1 mM dithiothreitol, and sodium azide (0.2 gIliter). The enzyme eluted in the second chro-matographic step, with DEAE-cellulose-Seph-adex A-50 columns, could be stored in thissolution at room temperature for 3 monthswith little loss of activity. In 0.1 M K+-Na+PO4buffer (pH 8.2), the enzyme showed maximalactivity at 57°C (Fig. 3); however, the opti-mum may be somewhat higher since the en-

zyme may not have been saturated with oxy-gen at this temperature.HP dioxygenase from B. stearothermophilus

attacked 3,4-dihydroxyphenylpropionic acidand DL-3,4-dihydroxymandelic acid in additionto 3,4-dihydroxyphenylacetic acid (homoproto-catechuic acid) (Table 3). Slight activity to-wards protocatechuate could be measured, butcompounds that were reported to be attackedby the HP dioxygenase from P. ovalis (15) atlow rates, including catechol, 4-methylcate-chol, L-3,4-dihydroxyphenylalanine, and L-do-pamine, were not oxidized. However, the en-

zyme from P. ovalis showed much greater ac-

tivity towards homoprotocatechuate than didHP dioxygenase from B. stearothermophilus;the limit of detection of activity for our assaysystem was about 0.3 nmol/min. The apparentK,,, for 3,4-dihydroxyphenylpropionate was

about the same as that for 3,4-dihydroxyphen-

.' 1.2

E

CL

4-

0

(.)

E 0.8

-0.

t .

U.0.2

cn

20 28 36 44 52 60 68TEMPERATURE, °C

FIG. 3. Effect oftemperature upon specific activityofHP dioxygenase from B. stearothermophilus. Theassay system contained (per milliliter): 98 pumol ofK+Na+PO4 (pH 8.2), 1 ,mol ofhomoprotocatechuate,and 11.4 pg ofenzyme. After temperature equilibra-tion, reactions were started by the addition of en-

zyme.

ylacetate, with about half the maximum ve-

locity, whereas the apparent K,,, for DL-3,4-dihydroxymandelate was about 10 timesgreater with little change in V,,,,,,. For proto-catechuate, the apparent K,,, was muchgreater and the V,,,,.. was small. Various com-pounds were tested as inhibitors. No effect wasobserved for potassium cyanide, sodium azide,and EDTA, each at a concentration of 2 mM;p-hydroxymercuribenzoate and dithiothreitolshowed about 13% inhibition at 0.1 mM; and0.1 mM ferrous ammonium sulfate inhibitedby 32%, although this effect was complicatedby a tendency of ferrous hydroxide to precipi-tate at the pH (8.2) of the determination.Thermodynamic properties. An Arrhenius

plot for log V,,,ax and 1T gave lines with differ-ent slopes intersecting at about 32°C (Fig. 4).

TABLE 3. Substrates oxidized by HP dioxygenase a

)~mAx Sp act

Compound (nm) of (,mol/minproduct per mg of

protein)b

3,4-Dihydroxyphenyla- 380 0.171cetic acid

DL-3,4-Dihydroxyman- 375 0.142delic acid

3,4-Dihydroxyphenyl- 380 0.099propionic acid

3,4-Dihydroxybenzoic 355 0.001acida No ring-fission product could be detected from:

3,4-dihydroxyphenylglycol, L-3,4-dihydroxyphenyl-alanine, L-dopamine, catechol, 4-methylcatechol,3,4,5-trihydroxyphenylacetic acid, 2,3,4-trihydroxy-phenylacetic acid, or 3,4-dihydroxycinnamic acid.bThe assay system at 29°C contained (per milli-

liter): 98 ,umol of K+-Na+PO4 buffer (pH 8.2), 0.1,umol of compound tested, and a suitable addition ofenzyme.

TABLE 4. Values of apparent K,m and Vma, forvarious substrates ofHP dioxygenase a

VmaxSubstrate K,, (;LM) (tmol/min

per mg)

3,4-Dihydroxyphenyla- 3.4 ± 0.2 0.196cetic acid

3,4-Dihydroxyphenyl- 3.7 ± 0.4 0.091propionic acid

DL-3,4-Dihydroxyman- 35 ± 3 0.225delic acid

3,4-Dihydroxybenzoic 450b 0.005bacida Experimental conditions as described in foot-

note b of Table 3, except that the temperature was32'C and substrate concentrations were varied.

bEstimated by the method of Eisenthal and Cor-nish-Bowden (6).

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HOMOPROTOCATECHUATE DIOXYGENASE 695

Enthalpies of activation (AHt) were approxi-mately 4.8 and 13 kcal/mol at temperaturesbelow and above the transition point, respec-tively. On the other hand, below the transitiontemperature, AS*: was -45 entropy units;above the transition temperature, ASt was -18entropy units. The free energy of activation(AGt) was essentially the same for each region(about 18 kcal/mol). Apparent Km values de-creased with increasing temperature to about320C and then increased (Table 5). Apparentfree energies of binding (-AG) increased from7.2 kcal/mol at 210C to 7.6 kcal/mol at 320C anddeclined slightly to 7.5 kcal/mol at 470C. Plotsof log Km against 1IT were not linear for thetwo regions above and below 320C, but tan-gents drawn to the curves at 28 and 380C gaveapparent enthalpies of binding (AH) of -5.4and +9.3 kcal/mol, respectively; the corre-

o \<~~~3 kcol/mol

0

o5kalm cl/mol

06-J

%_0

3.0 3.2 3.4I/T XI03

FIG. 4. Effect of temperature upon maxcimum re-action velocity of HP dioxgenase, expressed as anArrhenius plot.

TABLE 5. Effect of temperature on the apparent Kmfor homoprotocatechuatea

Temp (°C) Km (AM)21.0 4.7 + 0.128.0 4.0 + 0.130.5 3.4 ± 0.432.0 3.4 + 0.238.0 5.8 + 0.147.0 7.4 ± 0.1

a Experimental conditions as described in foot-note a of Table 4, except that temperatures werevaried.

sponding values of AS were 6.7 and 54 entropyunits.

DISCUSSIONHP dioxygenase of B. stearothermophilus

was purified to apparent homogeneity asjudged by its behavior in disc-gel electrophore-sis and by sedimentation equilibrium meas-urements. The average of molecular weightdetermined by three procedures was 106,000.Disc-gel electrophoresis in the presence ofSDSafter reduction with ,8-mercaptoethanolshowed one major protein band of molecularweight 33,000 to 35,000, and the same bandwas obtained without this treatment. Thesedata suggest that HP dioxygenase from B.stearothermophilus is a trimer composed ofidentical subunits without intersubunit cova-lent bonds. Although they are rather uncom-mon, trimeric protein molecules do occur (11).HP dioxygenase from P. ovalis is a tetramer(15). There are other differences also apparentbetween the P. ovalis and B. stearothermophi-lus enzymes. The specific activity of the puri-fied B. stearothermophilus enzyme was muchlower than that from P. ovalis (9) and ap-peared to lose activity progressively duringpurification procedures. The ultraviolet spec-trum of the B. stearothermophilus enzymeshowed X.,r at 275 nm and Xmi. at 247.5 nm,whereas that from P. ovalis had Xmaz at 280 nmand Xmin at 252 nm (15). There were also differ-ences in amino acid composition. The B. stea-rothermophilus enzyme has larger proportionsof tyrosine, phenylalanine, lysine, and glu-tamic acid than the P. ovalis enzyme, whereasthe reverse is true for glycine, alanine, histi-dine, valine, and methionine. Other differ-ences in properties were evident. Potassiumcyanide potently inhibited the enzyme from P.ovalis (9) but had no effect upon that from B.stearothermophilus; this enzyme was alsomuch less strongly inhibited by p-hydroxy-mercuribenzoate. HP dioxygenase from thebacillus was protected from inactivation byEDTA, a compound that inhibited the enzymefrom P. ovalis by 20% after preincubation for 2h (9). Activity lost by the latter enzyme onstorage could be regained on incubation withFe2+ ions (9), which inhibited the enzyme fromthe bacillus. The specific activity of this en-zyme for 3,4-dihydroxyphenylpropionate wasabout half of that for homoprotocatechuate,whereas the factor was about 0.03 for the en-zyme from P. ovalis (15).The Arrhenius plot for the B. stearother-

mophilus enzyme was discontinuous and con-cave upwards (Fig. 4), a feature that is rather

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unusual in enzyme-catalyzed reactions (5). Al-though breaks in Arrhenius plots of biologicaland enzymatic processes are now accepted,there is no general agreement as to the reasonsfor their occurrence (8). Massey (12) obtainedan upward bend in the Arrhenius plot of fuma-rate hydratase reaction in alkaline solutions.He suggested that this discontinuity might bedue to a dissociation of the enzyme into units ofsmaller molecular weight as the temperaturewas increased, catalysis by these smaller unitsrequiring a higher activation energy. It is notknown whether such a dissociation occurs inHP dioxygenase of B. stearothermophilus, butsome change of conformation appears to be in-dicated by the changes observed at 32°C in ap-parent K,, (Table 5) and also by increases inAHt. These increases are compensated by achange to a more favorable value of ASt athigher temperatures (from -45 to -18 entropyunits), leaving A Gt virtually unaltered.

It may be observed that upward curvaturesin Arrhenius plots, such as we found, cannot bedescribed as characteristic of thermophilic en-zymes. Such a plot for enolase from the extremethermophile Thermus aquaticus YT-1 ex-hibited a downward curvature, with lower acti-vation energy at higher temperatures (19). Cal-culations from the data given by Stellwagen etal. (19) show that the entropy of activation at50°C was more negative than at 20°C (about-47 compared with -9 entropy units). It ap-pears, therefore, that neither the direction ofcurvature of Arrhenius plots, nor the change inthe magnitude of ASt, can be correlatedgenerally with thermophilicity.The initial decrease in the apparent K,, for

homoprotocatechuate and its subsequent in-crease with temperature might be ascribed to achange in binding, with AH = -5.4 and +9.3kcal/mol for binding at 28 and 32°C, respec-tively. However, the plots of log K,, against 1Twere not linear, suggesting that K, did not, infact, approximate to a simple dissociation con-stant for binding; rather, it appeared that thechanges observed may have been due to differ-ent effects of changes in temperature upon thecomponent rate constants of K,,,. Of the sub-strates investigated, homoprotocatechuate hadthe lowest apparent K,,, and highest VX,,,,. Ex-tension of the side chain by an extra methylenegroup, as in 3,4-dihydroxyphenylpropionic acid,had little effect upon apparent K,,, (Table 4), butthis quantity was increased about 10-fold byintroducing a hydroxyl group (DL-3,4-dihydrox-ymandelate) and more than 100-fold by remov-ing the original methylene group (3,4-dihydrox-ybenzoate). These data suggest that a hydro-

phobic enzyme site is involved in binding; how-ever, involvement of carboxyl, as well as meth-ylene, is indicated by the observation that 4-methylcatechol was neither a substrate nor in-hibitor. V,,,,, was reduced by about one-halfwhen an extra methylene group was present,was slightly increased when a hydroxyl groupwas introduced, and was greatly reduced whenthe carboxymethyl group of homoprotocate-chuate was replaced by carboxyl.

ACKNOWLEDGMENTS

I thank Mark Elstad, who, when working in Peter Chap-man's laboratory, isolated and identified B. stearother-mophilus and showed that HP dioxygenase was induced inthis organism. I also thank Peter Chapman for a gift ofcrystalline 3,4-dihydroxyphenylpropionic acid, and StanleyDagley for many helpful discussions.

This investigation was supported by Public Health Ser-vice grant ES AI 00678 from the National Institute of Envi-ronmental Health Sciences.

LITERATURE CITED

1. Adachi, K., Y. Takeda, S. Senoh, and H. Kita. 1964.Metabolism of p-hydroxyphenylacetic acid in Pseu-domonas ovalis. Biochim. Biophys. Acta 93:483-493.

2. Blakley, E. R., H. Halvorson, and W. Kurz. 1967. Themicrobial production and some characteristics of8- carboxymethyl - a - hydroxymuconic semialdehyde.Can. J. Microbiol. 13:159-165.

3. Commission on Biochemical Nomenclature. 1972. En-zyme nomenclature, p. 104. Elsevier Publishing Co.,Amsterdam.

4. Davis, B. J. 1964. Disc electrophoresis. II. Method andapplication to human serum problems. Ann. N.Y.Acad. Sci. 121:404-427.

5. Dixon, M., and E. C. Webb. 1964. Enzymes, 2nd ed., p.159. Academic Press Inc., New York.

6. Eisenthal, R., and A. Cornish-Bowden. 1974. The di-rect linear plot. A new graphical procedure for esti-mating enzyme kinetic parameters. Biochem. J.139:715-720.

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8. Han, M. H. 1972. Non-linear Arrhenius plots in temper-ature-dependent kinetic studies of enzyme reactions.I. Single transition process. J. Theor. Biol. 35:543-568.

9. Kita, H. 1965. Crystallization and some properties of3,4-dihydroxyphenylacetate 2,3-oxygenase fromPseudormonas ovalis. J. Biochem. (Tokyo) 58:116-122.

10. Kita, H., M. Kamimoto, S. Senoh, T. Adachi, and Y.Takeda. 1965. Crystallization and some properties of3,4-dihydroxyphenylacetate-2,3-oxygenase. Biochem.Biophys. Res. Commun. 18:66-70.

11. Klotz, I. M., D. N. Darnall, and N. R. Langerman.1975. Quaternary structure of proteins, p. 294-402.In H. Neurath and L. R. Hill (ed.), The proteins, vol.1, 3rd ed. Academic Press Inc., New York.

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Errata

Purification and Properties of Homoprotocatechuate2,3-Dioxygenase from Bacillus stearothermophilus

MOIDEEN P. JAMALUDDINDepartment of Biochemistry, College of Biological Sciences, University of Minnesota,

St. Paul, Minnesota 55108

Volume 129, no. 2, p. 690, Abstract, line 10: "Ki" should read "Vma,."Page 692, Table 1, column 1, line 4: "DEAE-cellulose-Sephadex" should read "DEAE-Sephadex."Page 695, column 1, lines 18 and 19: "-5.4 and +9.3" should read "+5.4 and -9.3."Page 695, column 2, line 1: "6.7 and 54" should read "42 and -6."Page 696, column 1, line 40: "-5.4 and +9.3" should read "+5.4 and -9.3."Page 696, column 1, line 41: "32°C" should read "38C."

Lipophilic O-Antigens in Rhodospirillum tenueJ. WECKESSER, G. DREWS, R. INDIRA, AND H. MAYER

Institut fur Biologie II, Lehrstuhl fur Mikrobiologie der Universitat, and Max-Planck-Institut furImmunbiologie, D-7800 Freiburg i.Br., West Germany

Volume 130, no. 2, p. 631, Table 2, footnote a: Should read "Percentage of0-antigen dry weight."

362