Dehalogenase, from Pseudomonas sp.

0021-9193/82/050522-06$02.00/0

Purification and Properties of

a

New

Enzyme, DL-2-Haloacid

Dehalogenase, from Pseudomonas

sp.

KENZOMOTOSUGI,t NOBUYOSHI ESAKI,ANDKENJI SODA* Institute forChemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan

Received 21 September 1981/Accepted 23 December 1981

A

new enzyme,

DL-2-haloacid

dehalogenase,

was

isolated

and purified

to

homogeneity from the

cells

of Pseudomonas

sp. strain 113. This enzyme

catalyzed

non-stereospecific dehalogenation

of

both

of the

optical

isomers of

2-chloropropionate through

an

SN2

type

of

reaction;

L-and D-lactateswere

formed

from D-

and

L-2-chloropropionates,

respectively.

The enzyme

acted

on

2-halogenated aliphatic carboxylic acids whose

carbon chain

lengths

wereless than

five. It also

dehalogenated trichloroacetate

to

form oxalate

andshowed maximum

activity

at

pH 9.5. The Michaelis

constantsfor

substrates

were asfollows: 5.0 mM

for

monochloroacetate,

1.1 mM

for

L-2-chloropropionate,

and 4.8 mM for

D-2-chloropropionate. DL-2-Haloacid dehalogenase

wasinhibited

by

HgCl2, ZnSO4,

and

MnSO4,

butwasnotaffected

by

thiol reagents, suchas

p-chloromercuriben-zoate

and iodoacetamide.

Thisenzymehad a molecular

weight

of about

68,000

and

appeared

tobe

composed

oftwosubunits identicalinmolecular

weight.

2-Halogenated alkanoic

acids

are

toxic

to

mammals and

other

organisms

and have been

used as

herbicides

and

pesticides.

These

com-pounds

have

been

reported

to

be

decomposed

by bacterial

enzymes

called

dehalogenases

(10).

Two

kinds

of dehalogenases

have been

found,

haloacetate

dehalogenase

(EC 3.8.1.3)

and

2-haloacid

dehalogenase

(EC

3.8.1.2) (11).

The

former is further

classified

into

two

different

types; one

of

these

acts on

fluoroacetate

(6,

12,

20), and the

other

does

not

(14; J. I. Davis and

W.

C. Evance, Biochem J.

82:50p-51p, 1962).

Recently, the haloacetate

dehalogenase of

Mor-axella sp. was

found

to

be

determined

by

a

plasmid (13). The dehalogenases demonstrated

so

far act solely on

L-2-haloacids (7,

15).

Recently, we isolated

a

species

of

Pseudomo-nas

that

assimilates

D-2-chloropropionate

as a

sole carbon source and

suggested

the

occur-rence

of a new

dehalogenase which can act on

both

D-

and

L-2-haloacids (Motosugi,

Esaki, and

Soda, Arch. Microbiol., in press). In this paper

we

describe the purification of this enzyme from

bacterial

cells

and the

physicochemical and

en-zymological properties of its purified form.

MATERIALSAND METHODS

Materials. DEAE-cellulose was purchased from

Serva, hydroxyapatite was obtained from Seikagaku Kogyo, Tokyo, Japan, NAD was obtained from Kyowa Hakko Kogyo, Tokyo, Japan, D- and L-lactate

tPresent address: Research andDevelopmentCenter, Uni-tikaLtd., Uji, Kyoto-Fu 611, Japan.

dehydrogenases were obtained from Boehringer

Mannheim Gmbh, Mannheim, West Germany, and

Sephladex

G-150 was obtainedfrom Pharmacia Fine Chemicals, Uppsala, Sweden D- and

L-2-chloropro-pionic acids were prepared from D-alanine and L-alanine, respectively, bythemethod of Fuetal. (5).

Other chemicalswereofanalytical grade.

Conditionsforcellgrowth. Theisolation and charac-terization of Pseudomonas sp. strain 113 have been

described elsewhere (Motosugi etal., in press). The cells were grownaerobicallyat30°Cinmedium

con-taining 0.3%DL-2-chloropropionate,0.5% (NH4)2SO4,

0.1% KH2PO4, 0.1% Na2HPO4 12H2O, and 0.01% MgSO4-7H2O (pH 7.0). DL-2-Chloropropionate was sterilized separately by filtration through a membrane filter (type HA; pore size, 0.45 ,um; Millipore Corp.,

Bedford,Mass.).Cells were harvested by

centrifuga-tionatthe end of the logarithmic growthphase and stored at-20°Cafter they were washed twice with 50 mMpotassiumphosphatebuffer (pH 7.5).

Enzyme assay and analytical method. The enzyme wasassayed by determining the halogen ions released from the substrates. The standard assay mixture (1.0 ml) contained 25 Fmol ofDL-2-chloropropionate, 100 ,umol of Tris-sulfate buffer (pH 9.5), and enzyme. Afterincubation at 30°C for 10 min, the reaction was terminated by adding 0.1 ml of 3 N H2SO4. The chlorideions released were determined spectrophoto-metrically with mercuric thiocyanate and ferric ammo-nium sulfate (9). Bromide and iodide ions were also determined in the same way. One unit of enzyme

activity was defined as the amount of enzyme that

catalyzedthe dehalogenation of 1 ,umol of substrate per min. Protein was determined by the method of Lowry et al. (16), using bovine serum albumin as a standard. Formostcolumn fractions, the protein elu-tionpatternswereestimated by absorption at 280 nm. D-Lactate andL-lactate were determined with D- and 522

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NEW DEHALOGENASE FROM L-lactate dehydrogenases by themethodof Gutmann

andWahlefeld(8).

Enzymepurification. Alloperationswerecarried out at 0 to5PC,unlessotherwise specified.

(i) Step 1:cel extract. Cells (350 g, wet weight) were suspended in 1.0 liter of 50 mM potassium phosphate buffer (pH 7.5) and disrupted continuously with a

Dynomill (W. A. Bachofen) containing glass beads (diameter, 0.1 to 0.2 mm). The intact cells and cell debris were removed by centrifugation.

(ii)Step 2:protaminesulfate. To the cell-free extract (1.2 liters), a2.0%oprotamine sulfate solution (pH 6.5, 120 ml) was added slowly with stirring, and the precipitate was removed by centrifugation and dis-carded.

(iii) Step 3: ammonium sulfate fractionation. The

supernatant solution (1.2liters) was brought to 40%o

saturationwith ammonium sulfate, and theprecipitate wasremoved bycentrifugation and discarded. Ammo-nium sulfate was added to the supernatant solution (1.3 liters) to 70%o saturation. The precipitate was collected by centrifugation and dissolved in 50 mM potassium phosphate buffer (pH 7.5). The enzyme

solutionwasdialyzed overnightagainst the same

buff-er. The insoluble materials formed during dialysis wereremoved bycentrifugation.

(lv)

Step 4: DEAE-cellulose. The dialyzed enzyme solution (270 ml) was applied to a DEAE-cellulose

column (8 by 50 cm)equilibratedwith 50 mM

potassi-umphosphate buffer (pH 7.5). After the column was washed with 2 liters of the buffer, elution was carried outwith a lineargradient(800ml of 50 mMpotassium phosphatebuffer,pH 7.5, in themixingchamber and 800 mlof 0.5 M potassiumphosphatebuffer,pH7.5,in thereservoir; flow rate, 150 ml/h; each fraction, 15

ml). Theenzyme eluted at a bufferconcentration of

about 0.1 M. Active fractions wereconcentrated by

addingammonium sulfate (70%6 saturation) and then

dialyzed against 5 mM potassium phosphate buffer (pH7.5).

(v) Step 5:hydroxyapatite.Theenzyme solution was

placed onto a hydroxyapatite column (4 by 35 cm)

equilibrated with 5 mM potassium phosphate buffer (pH7.5).After the column was washed with 0.5liter of

thesame buffer, elution was carried out with a linear

gradient of potassium phosphate buffer(5 to100 mM in atotal volume of 1.0liter)at a flow rate of 50ml/h,and 5-ml fractions were collected. The active fractions, whicheluted at bufferconcentrations between 15 and 20 mM, were pooled and concentrated by adding ammonium sulfate

(70%o

saturation).

(vi) Step 6: gelMitration.Theenzymesolutionwas

applied toa Sephadex G-150 column (3 by 130cm)

equilibrated with 50 mMpotassium phosphatebuffer

(pH7.5) and then eluted with the buffer at a flowrate of 10 ml/h. Active fractions were concentrated by

ultrafiltration with a Diaflow membrane.

Polyacrylamide

gelelectrophoresis.Discgel

electro-phoresisin a7.5%polyacrylamide gelwasperformed

by the method of Davis (2). After electrophoresis, proteinwasstained with amidoblack,and theenzyme

activitywasstainedbyamodification of the method of Dietz and Lubrano(3). The extrudedgelsweresoaked in areaction mixturecontaining2.5 mlof 0.1 M Tris-sulfate buffer(pH9.0),1mlof 0.2 M DL-2-chloropro-pionate, 0.4 mlof NAD (30mg/ml), 2.5 ml ofNitro Blue Tetrazolium (1 mg/ml), 0.25 ml of phenazine

methosulfate(1mg/ml),andapproximately10 U of D-orL-lactatedehydrogenase(or bothenzymes)at

300C

for30 minin the dark.

Determination of molecular weight. The molecular weight of the subunits wasestimatedbysodium

dode-cyl sulfate (SDS)gelelectrophoresis, usingthemethod

of Weber andOsborn(21). Theenzyme wasdialyzed against10 mMsodiumphosphate buffer (pH 7.0)and thendenaturedbytreatment with a1% SDSsolution containing1% 2-mercaptoethanolat100°Cfor 5 min. The standard proteins, including the a, ,B, and 1' subunits of RNApolymerase

(Mr,

39,000,155,000,and 165,000), bovine serum albumin

(Mr,

68,000), and

trypsininhibitor

(Mr,

21,500), were treated in the same way. Ultracentrifugation was in a Spinco model E ultracentrifugeequippedwith a phase plate as a

schlie-ren diaphragm and a Rayleigh interference optical system. The topspeeds ofultracentrifugationfor the

sedimentation velocityexperimentand the sedimenta-tion equilibrium experiment were59,780 and 11,253

rpm,respectively.

Isoelectric focusing. Isoelectric focusing ofthe en-zyme in apolyacrylamide gelwas performedby the method ofRighetti and Drysdale (18), using carrier

ampholites in the pH range from 3.5 to 10.0 at4°C.

hie

(A)

..fli

a

6b;-K)

(B)

,L, .: ::.

_

_t .. ..M... ;.. !::' ':: :. :: :'::.. __ .e..: ,o!... :Bi.; ,: S :. L ;:: ...

FIG. 1. Disc gel electrophoresis of

purified

en-zyme. (A) Polyacrylamide gel electrophoresis. The

purified enzyme (30

F.g)

was applied to a 7.5% gel

column, which wasrun at pH 9.4. The direction of

electrophoresis was from the cathode (top) to the anode. (B) SDS-polyacrylamide gel electrophoresis.

Thepurifiedenzyme(10,ug)wastreated with 1% SDS at100I for 5 min andwas

electrophoresed

on a

10%o

gel containing 0.1% SDS. Protein was stained with Coomassie brilliant blue R-250. (C)

Cross-linking

of theenzyme withdimethylsuberimidate. The enzyme

(0.5mg/ml)wasincubated withdimethylsuberimidate

(2.5 mg/ml) in 0.2 M triethanolamine

hydrochloride

buffer(pH 8.5) at30°Cfor 12 h. The

protein (30 ,ug)

wastreated and

electrophoresed

asdescribed above

(B).

i VOL.150,1982

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TABLE 1. Purification of the enzyme Total Total S c il

Step protein activity

(U/mg)

a %) (mg) (U) Crude extract 34,200 20,500 0.6 100 Protamine treatment 8,420 16,000 1.9 78 Ammonium sulfate 3,430 12,000 3.5 59 DEAE-cellulose 810 8,200 10.1 40 Hydroxyapatite 240 5,100 21.0 25 SephadexG-150 104 3,700 35.7 18

Afterelectrophoresis,theslicedgelswereimmersedin water to measure the pH. Protein was stained with

Coomassie brilliant blue G-250.

Aminoacidanalysis. Aminoacidswereanalyzed by using the method ofSpackmanetal.(19)andaHitachi model 835 amino acid analyzer. The enzyme was hydrolyzed in 6 N HCIat110°Cfor12,24, 48,and72 h. Half-cystine was determined ascysteicacid after performic acid oxidationandhydrolysis (17). Trypto-phan and tryosineweredetermined spectrophotomet-rically by the method ofEdelhoch(4).

RESULTS

Enzyme

purification.

A summary of the en-zyme

purification procedure

is

presented

in

Ta-ble

1.

The

enzyme was

purified

approximately

60-fold

from the crudeextract of Pseudomonas

sp. strain

113,

and the overall

yield

was 18%.

The purified

enzyme showed a

single protein

band

upon

disc

gel

electrophoresis

and SDS

gel

electrophoresis (Fig. 1).

A

single

band

appeared

also

by

activity staining

atthe same

position

as

the

protein

band. The

homogeneity

of the en-zymewasalso demonstrated

by

ultracentrifuga-tion. The

enzyme sedimented as a

single

sym-metrical peak

during

the sedimentation

velocity

experiment,

and its sedimentation coefficient corrected to water at

20°C

was5.3S. The

puri-fied

enzyme could be stored at

-20°C

in the

presence

of

50%o glycerol

at least for 1 year

without

any

loss

of

activity.

Therefore,

the

enzyme was

routinely

storedat

-20°C

in 25 mM

potassium

phosphate

buffer

(pH 7.5)

containing

50%6

glycerol until it

was

used.

Molecular

weight

and subunit structure. The enzymehadamolecular

weight

of

approximate-ly 68,000,

as

determined

by sedimentation

equi-librium, assuming that the partial specific

vol-J. BACTERIOL. TABLE 2. Substratespecificity of the enzyme

Relative Substratel

activity

(%)b Monochloroacetate .. 33 Monobromoacetate ... 280 Monoiodoacetate...9 Dichloroacetate ... 8(4)c Trichloroacetate... 3

(1)d

D-2-Chloropropionate

. . 84

L-2-Chloropropionate

... 118

DL-2-Chloropropionate

..0...

DL-2-Bromopropionate

... 380 2,2-Dichloropropionate... 42(21)C

DL-2-Chloro-n-butyrate

. . 18

DL-2-Bromo-n-butyrate

... 220 DL-2-Bromo-n-valerate . . 18

aThe

following

substrates were inert:

monofluoro-acetate, chloroacetamide, chloroacetaldehyde,

3-chloropropionate, 2-chloro-iso-butyrate,and 2-chloro-n-caproate.

bThe initial velocitywas measured bydetermining

the halogens released for the first 10 min and is expressed as the relative activity compared with DL-2-chloropropionate (100%6). Eachreaction mixture(1.0

ml) contained 100 ,umol of Tris-sulfate buffer (pH9.5),

25,umol ofsubstrate, and 0.3 U of enzyme

(dichloro-acetate and trichloroacetate) or 0.06 U of enzyme (othersubstrates).

cValues areexpressed on the basis of the rates of

2-oxo acidrelease. The amount of chloride produced

was twice theamount of 2-oxoacid released. d Rate of oxalate release. The amount of chloride released was three times the amount of oxalate formed.

ume

of the

enzyme

is 0.74. The subunit

composition and the molecular weight of the

enzyme were

determined

by SDS gel

electro-phoresis. There

was a

single band of stained

protein

on

SDS

electrophoresis gels. The

molec-ular

weight of the subunit

was

estimated

to

be

about 35,000. This suggested that the

enzyme

has

a

dimer

structure.

When the

enzyme was

incubated with

a

cross-linking

reagent

(dimethyl

suberimidate) and then subjected

to

SDS

gel

electrophoresis, only

two

bands appeared (Fig.

1C).

The

lower-molecular-weight band

(Mr,

35,000)

was

the

monomer,

and the heavier

band

was

the

dimer

(Mr,

70,000).

Therefore,

the

en-zyme wascomposed

of

two

subunits

having

the same

molecular weight.

TABLE 3. StoichiometryofdehalogenationofD-andL-2-chloropropionates

Amtofsubstrate Amtofproductformed

(M.mol)'

Substrate that disappeared

(xmol) Chloride D-Lactate L-Lactate

L-2-Chloropropionate 10.0 10.2 9.3 0

D-2-Chloropropionate 10.0 9.9 0 10.5

aEach reaction was

carried

out at 30°Cfor 30minina reaction mixture (1.0 ml)containing 100 ,umol of Tris-sulfatebuffer (pH 9.5), 10

,umol

ofD-orL-2-chloropropionate,and 2.5 U of enzyme. A sample of each reaction mixture was analyzed for the chloride ions and lactateproducedandfor the substrate consumed.

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Substrate

specificity.

The

ability of the

enzyme to

catalyze the

dehalogenation of various

halo-gen

compounds

was

investigated, and the initial

velocities for

the substrates are shownin

Table

2. All

of the

monohaloacetates tested

except

monofluoroacetate

were

dehalogenated

effec-tively. The order of

reactivity

was as

follows:

bromoacetate

>

iodoacetate

>

chloroacetate.

Chloroacetamide and

chloroacetaldehyde

were not

substrates,

indicating that

a

free

carboxyl

group

is

necessary

for

a

compound

to

be

a

substrate. The halogen

must

be

at

C-2 for

a

compound

to

be

a

substrate;

3-chloropropionate

was not a

substrate.

2-Halopropionate

was

the

best

substrate,

followed by monohaloacetate,

2-halobutyrate, and

2-halovalerate in that order.

Higher homologs of 2-halovalerate

(e.g.,

2-chlorocaproate)

were

inert.

Both optical isomers

of

2-bromo-n-butyrate, 2-chloro-n-butyrate, and

2-bromo-n-valerate

were

substrates,

because

halogens

were also

released

stoichiometrically

from the racemic substrates

(see

below). This

is

compatible with

an

indiscriminate action of the

enzyme on D-

and

L-2-chloropropionates. The

Michaelis

constants

for the

substrates

were

de-termined

as

follows:

L-2-chloropropionate,

1.1

mM;

D-2-chloropropionate,

4.8 mM;

DL-2-chlo-ropropionate, 3.2

mM;

and monochloroacetate,

5.0

mM.

2-Haloacids

were

dehalogenated

to

the

corre-sponding 2-hydroxy acids, which

were

identified

by

gas

chromatography. The

stoichiometry of

the

dehalogenation of

D-

and

L-2-chloropropio-nateswas

investigated.

As

shown in

Table

3,

D-and

L-2-chloropropionates

were

converted

to

equimolar

amounts

of L-lactate and D-lactate,

respectively, and chloride. 2,2-Dihaloacids

were

dehalogenated

to

yield equimolar

amounts

of

2-oxo

analogs and twice molar

amounts

of

halo-gens;

glyoxylate and

pyruvatewere

formed from

dichloroacetate

and

2,2-dichloropropionate,

re-spectively. Trichloroacetate

was

converted

to

oxalate

and

chloride

(molar ratio, of 1:1:3).

Oxalate

was

identified by

paper

chromatogra-phy, using

phenol-water-formic

acid

(75:25:1,

vol/vol;

Rf,

0.61)

and

ether-formic acid-water

(5:2:1, vol/vol; Rf, 0.75).

Effect

of

pH and temperature.

Maximum

en-zyme

activity

was at

pH 9.5 when the

initial

velocities of halogen release from 25 mM

mono-chloroacetate,

DL-2-chloropropionate,

2,2-di-chloropropionate,

and

DL-2-chloro-n-butyrate

were

measured

at

30°C in

Britton-Robinson

buff-er

(1).

The enzyme was stable in the

pH

range

from

7

to

10

whenitwas

assayed

after

incuba-tion at

37°C for 10 min.

The initial

velocity

for thefirst

10

min

was

measured

with

DL-2-chloro-propionate

at

pH

9.5 and differenttemperatures. The maximum

activity

was observed at

45°C.

The enzyme was heated in 50 mM

potassium

phosphate

buffer (pH 7.5)

at

different

tempera-tures

for 15

min and retained

the

following

activities:

300C,

100%o;

35°C,

85%;

40°C, 55%;

and

450C, 30%.

Inhibitors.

Various

compounds

were

investi-gated for their inhibitory effects

on enzyme

activity

(Table 4). The

enzyme was inhibited

markedly by HgCl2 and ZnSO4.

When the

en-zyme was

incubated with

1 mM

HgCl2

in 50 mM

potassium phosphate buffer (pH 7.5)

at

30°C

for

15

min, it

was

inactivated completely.

However,

ZnSO4

did

not

inactivate the

enzymeunder the

same

conditions. Plots of reciprocals

of initial

velocities

versus

reciprocals

of

DL-2-chloropro-pionate

concentrations

at

several

fixed

concen-trations

of HgCl2

gave a

family

of

parallel

straight lines. This showed

that inhibition

by

HgC(2 is

not

competitive

withthe

substrate,

and

the

apparent

Ki

was

calculated

to

be

0.28 mM.

Double-reciprocal

plots of initial velocities

ver-sus

DL-2-chloropropionate concentrations

at

several fixed

concentrations of ZnSO4

gave a

group

of

straight lines intersecting

atthe

point

of

1/Km.

Thus, ZnSO4 is

a

noncompetitive inhibitor

for

the

enzyme,

with

anapparent

Ki

of

0.51

mM.

The

enzyme

activity

was

recovered partially by

EDTA; for

example, when

examined

in the

presence

of both 5 mM EDTA and

1

mM

inhibi-tor,

the

activities inhibited by 1

mM

HgC12

and 1 mM

ZnSO4

were

recovered

approximately

40

and

75%,

respectively, by 5

mM

EDTA.

The

enzyme was

little

affected by thiol

reagents.

TABLE 4. Effect ofinhibitors on enzyme

activity"

Reagentb

Inhibition

None...

0

LiSO4

... 2

MgSO4

... 10 Calcium lactate...

5

MnSO4

... 54

FeSO4.

... 8

CoSO4

... 9

NiSO4

... 43

CuS04

... 3

ZnSO4

... 92

AgNO3

... 36 Cadmiumacetate ... 2

BaSO4

... 6

HgCI2

... 94

Pb(N03)2

... 16 EDTA... 3

N-Ethylmaleimide

...

0

PCMBC... 0 lodoacetamide...

0

a The enzyme activity was measured by the stan-dard assay method.

IFinalconcentrations, 1.0mM. cPCMB,

p-Chloromercuribenzoate

(concentration,

0.01

mM).

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TABLE 5. Amino acidcompositionof theenzyme Content ~No.of

Aminoacid Content residuesper (mol%)

~~subunit'

Asparticacid 9.8 29 Threoninec 4.4 13 Serinec 3.8 11 Glutamic acid 10.5 32 Proline 6.0 18 Glycine 7.4 22 Alanine 12.7 38 Half-cystine 0.5 2 Valine 5.4 16 Methionine 1.2 4 Isoleucine 6.1 19 Leucine 9.6 29 Tyrosine 2.6 8 Phenylalanine 3.9 12 Lysine 4.6 14 Histidine 2.8 8 Arginine 6.4 19 Tryptophan 2.3 7

a Values are averages for 12-, 24-, 48-, and 72-h

hydrolyses.

bThe values were calculated on the basis of a

molecular weightof35,000.

cValues correctedto zerotime ofhydrolysis.

Amino acid

composition.

The amino acid

com-position of the enzyme

is shown in Table 5.The

predominant

residues

were

alanine,

glutamic

acid,

aspartic

acid,

and leucine.

The

isoelectric

point

of the enzyme

was

esti-mated to be pH 4.9

by isoelectric

focusing.

DISCUSSION

In

this

paper

we

show

that anew

dehalogen-ase

of

Pseudomonas sp.

actson

both

D-and

L-isomers of 2-haloacids

indiscriminately;

we

named this

enzyme

DL-2-haloacid

dehalogenase.

For

example,

D-

and

L-2-chloropropionates

are

dehalogenated

with

inversion

of

optical

config-uration

as

follows:

D(L)-CH3CHClCOOH

+

OH---

L(D)-CH3CHOHCOOH

+

Cl-The

enzyme is similar to the

known 2-haloacid

dehalogenase

(EC

3.8.1.2)

with

respect

to

sub-strate

specificity, but it is

distinctly

different in

stereospecificity.

All

enzymes studied so far that

work on the chiral

carbon of substrates act on

the

exclusive

enantiomers,

except for the

race-mases, such as arginine racemase (22).

Several of the 2-haloacid

dehalogenases

which

have

been described differ from each other in

substrate

specificity

(7,

15),

and none

of them

acts on

higher homologs

of

2-halobutyrate.

However, DL-2-haloacid

dehalogenase

dehalo-genates

2-bromo-n-valerate;

it is

unique

in this

J. BACTERIOL. respect.

Moreover, this

enzyme

releases all

of

the

chlorine

atoms

of

trichloroacetate

to

pro-duce oxalate. This is the

first

report

of

enzymat-ic

decomposition

of

trichloroacetate.

The

2-haloacid

dehalogenases studied

so

far have

a

broad

substrate

specificity,

but

none

of them

eliminates

fluorine from

2-fluoro

fatty acids.

Monofluoroacetate

wasnot

susceptible

to

DL-2-haloacid

dehalogenase.

2-Bromoacids

are

better

substrates

than the

corresponding 2-chloroacids.

This

is

compatible

with

the order of

magnitude

of bond

dissociation

energy

between carbon and

halogen.

Dehalogen-ation of

monoiodoacetate proceeded

much

more

slowly

than

dehalogenation

of the

corresponding

chlorine and

bromine

substituents

despite

the

small

bond

dissociation

energy

between carbon

and

iodine. The

bulkiness

of iodine

probably

prevents

monoiodoacetate from

binding

to

the

active site of the

enzyme.

ACKNOWLEDGMENTS

Wethank A. Hanawa and H.Nara,Research and Develop-mentCenter,UnitikaLtd., Kyoto, Japan, forencouragement.

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