Optical activity and solvent perturbation of fumarase

20 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

36001

OPTICAL A C T I V I T Y AND SOLVENT P E R T U R B A T I O N OF FUMARASE

M A R G A R E T E. S O R E N S E N AND T H E O D O R E

T. H E R S K O V I T S *

Department of Chemistry, Fordham University, New Yorh, N.Y., zo458 (U.S.A.) ( R e c e i v e d A u g u s t 2nd, 1971)

SUMMARY

The circular dichroism (CD), optical rotatory dispersion (ORD), and the solvent perturbation difference spectra of fumarase in the far- and near-ultraviolet region have been investigated. The CD spectrum of fumarase is characterized by CD bands centering at 222, 209, and 193 nm with I01a = --18 700, --18 800, and 45 500 degrees.cm 2-dmole -1, respectively. The corresponding ORD spectrum has a trough and m a x i m u m at 233 and 198-199 nm, with [m'la = --6880 and 34 9 °0 degrees .cm 2" dmole 1. In the aromatic absorption region the CD spectrum is dominated by tryptophan contributions with a slightly negative band at about 300 nm and positive shoulders and maxima at 291-292, 283, and 275-278 nm, with I0128a = 30 degrees. cm 2.dmole 1. Phenylalanine contributions to the CD spectrum are observed at 259261 and 265-268 nm. The analysis of the CD and ORD spectra based on the published model parameters for a-, fl-, and random structures suggests the presence of approx. 50% a-helix, lO-3O% fl-structure, with the remaining portions of the enzyme in an unordered conformation. Inhibitors of fumarase were found to have little or no effect on the far-ultraviolet CD and ORD spectra. Interaction of the enzyme with malonate, suecinate, glutarate, or adipate were found to cause a slight enhancement of the CD spectrum in the aromatic region. The solvent perturbation difference spectra of fumarase obtained with 20% ethylene glycol, glycerol, and 50% deuterium oxide as perturbants indicate that 4-6 of the 8 tryptophyls and 20-24 of the 40 tyrosyls occupy surface positions where they are accessible to solvent, with the remaining groups being buried in the interior folds of the enzyme.

INTRODUCTION

Fumarase (EC 4.2.1.2) catalyzes the reversible hydration and dehydration of fumarate to L-malatel, ~. The optical rotatory dispersion (ORD) and the related Mof" To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

Biochim. Bi@hys. Acta, 257 (1972) 2o 29

OPTICAL ACTIVITYOF FUMARASE

21

fitt-Yang parameters of this important enzyme of the Krebs cycle have been reported 8-~. These investigations have suggested the presence of about 5o% a-helical structure in the native enzymO, 4. We have extended the ORD studies into the farultraviolet region below 2oo nm, and have also carried out a detailed examination of the circular dichroism (CD) in both the far-ultraviolet, peptide absorbing region and the near-ultraviolet, aromatic region of the enzyme. This communication presents the results and analysis of our findings together with a study of the solvent perturbation behavior of the.tryptophyl and tyrosyl residues in fumarase, related to the near-ultraviolet CD studies. The effects of some of the inhibitors of the enzyme on the ORD, CD, and solvenf perturbation difference spectra are also reported in this paper. MATERIALS AND METHODS

Reagents L-Malic, D-malie, adipic, glutaric and citric acids were purchased from Mann Research Laboratories, whereas malonie and succinic acids were obtained from Fisher Scientific Co. With the exception of adipic acid, which was recrystallized from water, all of these acids were recrystallized from anhydrous diethyl ether after being refluxed for 30 h (ref. 6). As required, weighed quantities of crystals were dissolved in distilled water, neutralized with N a O H and adjusted to pH 7.3. All other reagents and solvents used were of the purest commercially available quality or spectroscopic grade and were used without further purification. The water used was distilled in an all-glass still. Fumarase Crystalline fumarase was prepared from swine heart muscle according to the procedure described by KANAREK AND HILL3 or obtained from Boehringer and Mannheim Corp. and Sigma Chemical Corp. These enzyme preparations were further purified b y chromatography on Bio-gel 300 columns at 4 ± I° (Fig. i). We have found that the specific activities of both our and the commercial enzyme preparations could be substantially increased by chromatography (Fig. I). Initially, the observed specific activities ranged from 16 45 ° to 31 700 units per rag, whereas the chromatographically purified samples had improved activites of 32 200-33 IOO activity units per mg of enzyme. Correspondingly, the mean residue rotation, Em']~ at 233 nm of the enzyme preparations were found to change from initially --3000 to --529 ° to --620o to --7155 degrees, cm 2. dmole -a. The data of Fig. I indicates clearly that the less active enzyme fractions have also less negative Em'lx values. The fumarase samples used for the measurements reported in this paper have exhibited specific activities of 30 600-33 IOO units/rag enzyme. Enzyme activity and concentration Fumarase was assayed by a modification of the method of FRIEDEZ~ et alY described by KANAREK AND HILL3. Activities were determined spectrophotometrically at 250 nm in stoppered cuvettes with r.o-cm light path using o.05 M L-malate as substrate, in solutions containing 0.05 M sodium phosphate buffer (pH 7-3) and o-7 ° volume percent of ethylene glycol, glycerol, or deuterium oxide. Protein concentrations were based on the extinction coefficient of o.51 per mg of enzyme per ml of soBiochim. Biophys. Acta, 257 (1972) 2o-29

22

M.E.

SORENSEN, T. T. HERSKOVITS

---7 B

•0 x 25

30

-6 -5 'o ,.4 x -3

•~ 10 o. ~

-2

5 0

791113

3

$

E 0.7[

II

-1

9 11 13

C

N

; o.s~O.4,

~

O.20.10

• 0

4 6 10 12 Fraction Number

14

16

Fig. t. C h r o m a t o g r a p h i c purification of f u m a r a s e on a Bio-gel P-3oo (1. 5 cm × 24 cm) h a v i n g a 2-cm layer of Bio-gel P-6o at the top and b o t t o m of the column. The column was equilibrated with O.Ol M sodium p h o s p h a t e buffer (pH 7.3) at 4 ± I°, which was also used as the eluent. Fractions of 3 ml were collected at flow rates of 13 15 llll/h. The u p p e r A and B p a r t s of this figure give the specific activities and m e a n residue rotation, Ern'i2aaof one of the enzyme p r e p a r a t i o n s used. The lower C p a r t of the figure gives the a b s o r b a n c e of the fractions collected. The fractions with the highest specific activity (Fractions 4-8) were usually combined and used for the studies reported.

lution at 28o nm corrected for t u r b i t y a. The latter correction was made b y subtracting the observed absorbance at 330 n m from the absorbance at 280 nm (ref. 3). Based on the molecular weight of 194 ooo (ref. 8), this value corresponds to the molar extinction coefficient of 9.89.1o 4.

ORD, CD and difference spectral measurements A Cary-6o recording spectropolarimeter equipped with a Cary-6oo2 circular dichroisrn a t t a c h e d was used for the O R D and CD measurements. Base lines or "zero" lines were run routinely with both solvent and the particular inhibitor used. Where found to be present, the contributions of the inhibitor to the O R D or CD spectra were subtracted from the enzyme curves. The O R D data is reported as mean residue rotation, [m'~~, corrected for the refractive index of the solvent 9, while the CD data is given in terms of the mean residue ellipticity, [01z. The latter were not corrected for solvent refractive index. The Moffitt-Yang a0 and b0 parameters 9 were based on least-square analyzed 313-578-nm dispersion data, with the dispersion constant, 4o, taken as 212 nm. For these calculations the mean residue molecular weight, M0, of IiO was used a. Difference spectral measurements were made in a Cary-I4 recording spectrop h o t o m e t e r equipped with a Io-fold absorbance scale expander attachment. Matched 4.5o-cm cylindrical double cells were used 1°. Solutions were prepared by 2-fold voluBiochim. Biophys. Acta, 257 (1972) 20-29

OPTICAL ACTIVITY OF FUMARASE

23

metric dilution1°, n, using the chromatographed enzyme solutions in o.oi or 0.02 M phosphate buffer (pH 7.3) and 40% ethylene glycol, glycerol or 99.88% deuterium oxide and o.oi M buffer or water. In order to minimize the possible effects of temperature and surface denaturation as a result of mixing and manipulation of the enzyme solutions, dilutions were made using precooled enzyme, perturbant, and buffer solutions, kept in an ice bath. Difference spectral measurements were made after the mixed solutions had reached room temperature. After the spectra were recorded, one part per IOO volumetric additions of o.I M buffered inhibitor at p H 7.3 were added to both the reference solution and the solution containing the 20% perturbants. After thorough mixing and equilibration of the enzyme solutions the difference spectra were rerecorded. Solvent perturbation difference spectra are reported in terms of the molar absorbance difference, d eM, calculated by dividing the observed absorbance or absorbance difference, AA, by the product of the enzyme concentration (in M) and the path length of the cell, 4.5 cm. All our data was analyzed using the curve fitting procedures and the model compound data of HERSKOVlTS AND S O R E N SEN12,13. Estimates of the apparent number of exposed tryptophyl and tyrosyl residues were obtained by fitting the enzyme curves according to the equation: Je~, ( e n z y m e ) = a.de~ ( T r p ) + b.Ae~ ( T y r )

(i)

where a represents the number of exposed tryptophyl residues, b the number of tyrosyls, z] ez (Trp) and zlex (Tyr), respectively, the molar absorbance difference values of free t r y p t o p h a n and tyrosine as a function of wavelength.

RESULTS AND DISCUSSION

CD and ORD results in the polypeptide absorbing region Native fumarase exhibits relatively large negative ellipticities with CD bands centering at 222, 209 and 193 nm. These three bands have mean residue ellipticities, [01~, of --18 700, --18 80o and 45 5 °0 deg rees'cm2"dmole-1, respectively. The ORD trough at 233 nm and the m a x i m u m at 198-199 nm have corresponding mean residue rotations, [m' 1~, of --6880 and 34 9 °0 degrees, cm 2. dmole -1. Fig. 2 and Table I TABLE THE

I

ORD

AND

CD

OF FUMARASE

Solvent, o.oi M phosphate

Inhibitor (r raM)

None Succinate Malonate Glutarate Adipate D-Malate Citrate

AND

THE

EFFECTS

OF SOME INHIBITORS

b u f f e r ( p H 7.3)-

ORD (degrees.cm 2. dmole 1)

CD (degrees. cm 2. dmole -1)

?m'],~.~

[m'has-~9

[0J~22

[0.72o9

[0J~93

-- 6880 ~-- 33 ° --68oo --6820 --67oo --670o -- 7000 --670o

34 9 0 0 2000 33 IOO 33 5 ° 0 34 4 °0 34 4 o 0 . . 33 i o o

- - 18 7 0 0 ± iooo --17 800 --18 200 --17 800 --18 ioo . . --t 7 900

- - 18 8 0 0 ± IOOO --17 400 --17 900 --17 80o --17 900

45 5 ° 0 J2 5 ° 0 0 40 2oo 41 7 0 0 38 7 0 0 4° 5o0

--17 30o

*

i

* Not measured because of the high rotation of the inhibitor at these wavelengths.

Biochim. Biophys. Acta, 2 5 7 ( I 9 7 2 ) 2 0 - 2 9

24

M. E. S O R E N S E N , T. T. H E R S K O V I T S

present a comparison and summary of the ORD and CD data obtained in this study. Our average Lrn'12a3 value of --688o ~ 33o based on the average of four different chromatographically purified enzyme preparations agrees closely with the values of --6925 and --665o degrees.cm 2.dmole 1 reported by BRADSHAWeL al. 4 and TEIPEL AND KOSHLAND5, suggesting the presence of substantial helical organization. The fit of our CD and ORD data in the far-ultraviolet I9O-25o-nm region with the polylysine data of FASMAN and co-workers 14,15, shown in Figs. 3A and 3B, suggests the presence of approx. 5of)o a-helix, 2o-3o(~,,;) fi-structure, with the remaining segments of the enzyme folded in an unordered or random form. The parameters of SAXENA AND WETLAUFEI~16, based on the CD spectra and X-ray diffraction data of three known proteins, suggests the presence of about the same amount of a-helix, with perhaps a lower estimate of fl-structure of the order of lO 2OO,o. Our estimates of helix content are in good agreement with the value of about 5o% a-helix* reported by KA:,AREK AND H I L L 3, based on the Moffltt-Yang b0 parameter of --311. The least-square analysis of ORD data in the 313-578-nm region gave, however, a somewhat lower

iI

40 CD 30

T~ ° 20

x~

lO

40 ORD

~

,

30

I~ --~

__~

o

20 .~ ?o

lO

-

o

- lO

-lO

,

200

,

220

h

240

i

260

I

J

200

,

i

220

A. in n m

,

i

240

,

r

,

260

,.X. in nrn

Fig. 2. T h e f a r - u l t r a v i o l e t C D a n d O R D s p e c t r a o f f u m a r a s e a n d t h e effects o f i m M g l u t a r a t e a u d a d i p a t e o n t h e s e s p e c t r a . - - - - - - - , f u m a r a s e a l o n e ; - - - - , f u m a r a s e in t h e p r e s e n c e o f i m M glut a r a t e ; . . . . , f u m a r a s e in t h e p r e s e n c e o f I m M a d i p a t e . All t h e e n z y m e s o l u t i o n s w e r e b u f f e r e d b y o . o i M s o d i u m p h o s p h a t e b u f f e r ( p H 7.3). T h e e n z y m e c o n c e n t r a t i o n s u s e d r a n g e d f r o m o.o 5 t o o.145 m g / m l . Cells o f o p t i c a l p a t h o f o. i o - i . o c m w e r e used.

b0 value of --267 (ref. 18) and a correspondingly lower estimate of helix content of 430/) *. The a0 value obtained was --80. Substrate and inhibitors are known to stabilize fumarasel:, 18. At the levels of 1-5o mM matonate and succinate were found to have essentially no effect on the ORD parameters and spectra of fumarase (Table II and Fig. 2B). Similarly, no significant effects on the ORD and CD spectra were observed using I mM glutarate, adipate or citrate as inhibitors (Table 1). The data obtained with these inhibitors 1 (Fig. 2 and Tables I and II) indicates that inhibition produces no gross changes in folding and chain conformation of the enzyme. * T h e s e e s t i m a t e s a r e b a s e d on t h e b o v a l u e of - - 6 3 o for too<)o a - h e l i x a n d O for b o t h t h e fl- a n d r a n d o m c o n f o r m a t i o n s 1 %

Biochim. Biophys. Acta, 257 (1972) 2 0 - 2 9

25

OPTICAL ACTIVITY OF FUMARASE .

,

,

,

,

40.

,

,

A

~

30

f\

40

B

30

CD

" ~t

ORD

......... .~,30~,2~

2o,~

~_~.~2°.i 1 ............... ~.,2o~,,3o~;

1o-~

°lj

o -lO

-10

m

200

220

240 ~. in nm

' 260

2~ '21o' ~o ' ~o

-20

A. in nm

Fig. 3. Comparison of the experimental and calculated CD and ORD spectra of fumarase based on the polylysine data of FASMANand coworkers14,1~. , fumarase in o.oi M sodium phosphate buffer (pH 7-3); - - ' - - , calculated curve for 50% a-helix, 30% /5-structure, and 20% random conformation; . . . . . . , calculated curve for 50% a-helix, 20% /~-structure and 30% random conformation. TABLE II THE

EFFECTS

FUMARASE

OF

INHIBITOR

CONCENTRATION

ON

THE

OPTICAL

ROTATION

AT 233

nm OF TWO

PREPARATIONS

Solvent, o.oi M phosphate buffer (pH 7.3). Inhibitor conch. (raM)

None I 5 12.5 25 37.5 5°

[m'7~03 (degrees.era 2. dmole) Succinate

Malonate

--6965 --6965 --7 °80 --7o8o --7175 --7 o8o --7080

-- 7155 --7o5 ° --705 ° -- 71oo --705 ° --7 loo --7155

CD spectra in the near-ultraviolet region The CD spectrum of fumarase in the aromatic absorbing region, above 25o nm is shown in Fig. 4 (data represented by the solid line). The spectrum exhibits a small negative dichroic band centering at about 300 nm, a positive band at 283 nm with E01283 = 30 degrees.cm 2-dmole -1, and small positive shoulders at 291-292 and 275278 nm that m a y be assigned to tryptophan transitions2°m. The m a x i m a and minima at 259-261 and 265-268 nm are characteristic of phenylalanine transitions~°,22m. T h e f a c t t h a t t h e d i c h r o i c b a n d s a r e n o t v e r y c l e a r l y r e s o l v e d in t h e p e a k r e g i o n a n d at 29~-292 n m suggests the effects of b r o a d e n i n g of the s p e c t r u m due to variations in t h e m i c r o e n v i r o n m e n t s o f t h e c h r o m o p h o r i e r e s i d u e s a n d p o s s i b l e o v e r l a p p i n g c o n t r i b u t i o n s o f t h e t y r o s y l g r o u p s . F u m a r a s e c o n t a i n s 8 t r y p t o p h y l , 40 t y r o s y l a n d 63 p h e n y l a n y l r e s i d u e s p e r m o l e o f e n z y m e , h a v i n g a m o l e c u l a r w e i g h t o f 194 ooo Biochim. Biophys. Acta, 257 (1972) 20-29

26

M. t';. SORENSEN, T. T. HERSKOVITS

(ref. 3). The solvent perturbation studies, described in the next section below, indicate that about 2- 4 of the tryptophyl and 16-2o of the tyroysl residues are buried with the remaining groups occupying surface positions where they are accessible to solvent. The polarity in the environments of the internal groups must be clearly different from those of the surface groups. Moreover, differences in the environment of the surface groups due to variations in the neighboring amino acid side chains and charged surface groups relative to any given group are bound to be present. The dicarboxylate inhibitors, malonate, succinate, glutarate and adipate were found to cause a slight increase in the intensity of the 283 nm as well as the neighboring CD bands of the enzyme. The effects of these inhibitors on the near-ultraviolet CD spectrum of fumarase are shown in Fig. 4 (data represented by the dashed, dotted i

4O .2-.-.

;,;T IeI~

%

""

i

i'1¢~[.4 0

,, li ! \'li

2~o 2~o

28o 2~o 3;0

31o

A. in n m

Fig. 4. The near-ultraviole CD spectra of f u m a r a s e ( ) and the effects of i mM succinate ( - - . - - ) , m a l o n a t e ( . . . . . . ), glutarate ( --), and adipate ( . . . . . . . . ). All the enzyme solutions were buffered b y o.oi M sodium p h o s p h a t e buffer (pH 7.3). The protein concentrations used r a n g e d from o.63 to o.66 mg/ml. Spectra were scanned at low speeds (o.2 to a b o u t 2 .~/sec.) with time c o n s t a n t setting of 3 or lO sec and a period setting of I.O. Cells of 5.o-cm p a t h length were used.

and dashed-dotted lines). The observed effects of inhibition are somewhat analogous to the effects of N-acetyl-D-glucosamine on the CD spectrum of lysozyme in this spectral region 24. Since no net burial of tryptophyl and tyrosyl groups are found with I mM succinate as inhibitor (Fig. 5B), it would appear that the effects of the dicarboxylate ions on the CD spectra of fumarase are due to changes in the charge distribution in the vicinity of the active sites of the enzyme or perhaps due to very subtle changes in the immediate vicinity of the active sites that affects the polarity and microenvironment of certain optically active chromophoric residues.

Solvent perturbation studies The solvent perturbation difference spectra of fumarase obtained with 2o% ethylene glycol and glycerol as perturbants are shown in Figs. 5 A and 5B. Fitting the enzyme data with the model parameters of free tryptophan and tyrosine12,13, Biochim.

Biophys.

A c t a , 257 (1972) 20-29

27

OPTICAL ACTIVITY OF FUMARASE

"°t ,.oI

lI"[ /.\

.~

260

h

2 0

i

1],.o "°

i

280 290 300 ~. in m n

310

260

270

2 8 0 290 ~,- in m n

300

310

Fig. 5. S o l v e n t p e r t u r b a t i o n difference s p e c t r a of f u m a r a s e o b t a i n e d with 2o% e t h y l e n e glycol (A) a n d 20% glycerol (B) as p e r t u r b a n t . T h e d a t a g i v e n b y t h e solid lines r e p r e s e n t t h e e n z y m e c u r v e s while t h e c u r v e s d r a w n w i t h t h e d o t t e d lines r e p r e s e n t t h e c a l c u l a t e d c u r v e s b a s e d on E q n . i giving t h e best fitting a a n d b p a r a m e t e r s . As i n d i c a t e d in t h e figure t h e d a t a were fitted w i t h 4 - 6 e x p o s e d t r y p t o p h y l a n d 20-24 e x p o s e d t y r o s y l residues. T h e c u r v e r e p r e s e n t e d b y t h e d a s h e d line in Fig. B w a s o b t a i n e d u s i n g I m M s u c c i n a t e inhibitor. All t h e e n z y m e solutions were buffered w i t h o.oi M s o d i u m p h o s p h a t e buffer (pH 7.3). T h e e n z y m e c o n c e n t r a t i o n s r a n g e d f r o m o. 5 to 0.78 m g ! m l . Cylindrical double cells of 4.5o-cm optical p a t h were used.

1.0 0.8

o.6 g

0.4 0.2

o

.~_

o t.0 %lycerol

0.8 ~

0.6

"~

0.4

i

0.2

._~

i

,

,

,

,

,

.

,

.

,

c x _ t .

. n t ,

.

,

D e u t e r i u m oxide

0.8

a,2'o',,/o 6 o ~ Volume per cent

Fig. 6. T h e effects of e t h y l e n e glycol, glycerol, a n d d e u t e r i u m oxide on t h e e n z y m i c a c t i v i t y a n d m e a n residue r o t a t i o n , En'J23.~,o f f u m a r a s e . T h e e n z y m i c a c t i v i t y a n d optical r o t a t i o n are e x p r e s s e d relative to t h e a q u e o u s v a l u e s o b t a i n e d in t h e a b s e n c e of t h e n o n a q u e o u s c o m p o n e n t or d e u t e r i u m oxide.

Biochim. Biophys. Acta, 257 (1972) 2o-29

2~

M. E. S O R E N S E N , T. T. H E R S K O V 1 T S

indicates that 4-6 of tile 8 tryptophyl residues and 2o-24 of the 4 ° tyrosyls are exposed to the perturbing influence of ethylene glycol and glycerol. These residues should occupy surface positions oll the enzyme with the remaining groups being buried in the interior regions where they are shielded from solvent access. Tile best fit of the enzyme data with different combination of exposed tryptophyl and tyrosyl residues are represented by the dotted and dash-dotted lines of this figure. We have also made measurements with 50% deuterium oxide as perturbant. While somewhat less reliable and more difficult to interpret because of the small and overlapping difference spectra usually produced by this perturbant12, la, about the same number of tryptophyls and tyrosyls were estimated to be exposed (i.e. 6 tryptophyls and 24 tyrosylslS). Since the enzyme conformation m a y be altered by the perturbants used, we have examined both tile ORD spectra and the enzymic activity of fumarase as a function of perturbant concentration. The data of Fig. 6 indices that neither the mean residue rotation at 233 nm nor the activity of fumarase is adversely affected at the levels of 2O°,o ethylene glycol, glycerol, and 5o% deuterium oxide used in our per turbation experiments. It is significant that neither glycol nor glycerol have any noticeable effects on the [m']2aa at relatively high concentrations (above 2o volume percent) where the enzymic activity is found to decrease. The fact that the im']2aa remains unaltered as a result of both solvent changes and inhibition by means of 1-5o mM succinate and malonate (Table II) suggests that fumarase has a comparatively rigid secondary and perhaps also tertiary structural organization. Inhibition of the enzyme by i mM succinate (Fig. 5B) is also found to have essentially no effect on the exposure of tryptophyl and tyrosyl groups. The loss of enzymic activity at high glycol and glycerol concentrations m a y be attributed to dissociation of the four subunits of the enzyme or perhaps to subtle conformational changes in the active site regions. While major changes in the structure and folding of the enzyme are unlikely in view of the fact that the optical rotation remains essentially unaltered, molecular weight and hydrodynamic measurements would be required to determine what exact effects these solvents have on the quaternary structure and non-helical regions of the enzyme. ACKNOWLEDGEMENTS

This investigation was supported by Grant GM 14468 from the National Institute of Health, U.S. Public Health Service and a Faculty Grant from Fordham University. Margaret E. Sorensen was a recipient of a Arthur J. Schmitt Predoctoral Fellowship in 1967 and the Vassie James Hill Fellowship of the American Association of University Women in 1969 . Part of this work was taken from her thesis submitted in partial fulfillment of the requirement for the Ph. D. degree, Fordham University, 1972. We are grateful to Professor D. D. Clarke for valuable suggestions and discussions throughout the course of our studies. REFERENCES i V. MASSEY, Biochem. J., 55 (1953) 172. 2 R . A. ALBERTY, i n P. D . BOYER, H . LARDY AND I~. MYRBACK, The Enzymes, V o l . 5, A c a d e m i c P r e s s , N e w Y o r k , 2 n d e d . , 1961 , p. 5 3 1 .

Biochim. Biophys. Acta, 2 5 7 (1972) 2 o - 2 9

OPTICAL ACTIVITY OF FUMARASE

29

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