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Nature of the coenzyme binding site of bovine malate dehydrogenase
SHORT COMMUNICATIONS
38I.
accord avec la constatation fare par HANSEN. FREELAND ET SCOTT9 dans le foie de poulet soumis it un r6gime riche en galactose. Mais ces auteurs n'ont pas fourni de donn6es quantitatives concernant les deux UDP-hexoses. Les rfisultats du dosage que nous avons effectu6 (Tableau I) nous ont r6v~16 une r6duction de l'UDP-glucose g6n6ralement de l'ordre de 5o°/. et un accroissement de l'UDP-galactose du quintuple ou plus. Ce ph6nom~,ne nous indique clue dans le cristallin in vivo, la production de TABLEAU
II
TENF,UR I';N GLYCOGI~'NE I)l'~ CRISTALLINS DI~ RATS ~G~S D|t 7 SEMAINF.S SOUMIS 2~. UN R~GIME ~ BASE DIe GLUCOSE OU DE GAI.ACTOSb; PENDANT ~ JOURS
die,~gitl,,, Glucose Galactost~
Expt. t
Expt, ~
Expt. 3
4~ 2o
46 2!
3° t5
l'UDP-galactose h partir du galactose x-phosphate + UTP est plus rapide que l'6pim~risation de l'UDP-galactose donnant de l'UDP-glucose. I1 en r~sulte une accumulation d'UDP-galactose avec r&luction tie l'UDP-glucose chez les rats soumis/t un r~gime fiche en galactose. De plus la eompftition du galactose I-phosphate et du glucose x-phosphate pour la formation ties UDP-hexoses par l'uridyltransf6rase semble ~galement intervenir. On en trouve les r6percussions dans la baisse du taux de l'UDP-glucose et du glycogtXne du cristallin (Tableau II). Ainsi it c6t6 de l'accumulation du galactose x-phusphate signal6e par SCHWARZ~T GOLBERC;~ une autre perturbation m6tabolique cons6cutive it la surcharge du r6gime riche en galactose a pu ~tre d6cel~e. I1 serait 6.videmment int6ressant de rechercher le mt~me ph6nom~-ne dabs les galactost~.mies par "erreur m6tabolique" con#nitale. lnstitut de Chimie Biologique, Facuit~ de Mddecine, Strasbour~ (France)
J. KI.~Tm P. MANDEL
I p. MANDEL I~T J . KLI~THI, liiochim, ltiophys...tcta, z8 (I958) 199. I V. SCItWARZ ~T L. GOLBF.RO, ltiochim, ltiophys...hta, t8 (1055) 310. a L. MANDRL, M. Wt.~TZVmTH, M. JACOR, M. P~:aRV V:T P, MANDt.:L, Compt. trod. soc. biol., t51. (1957) 993. 4 W. E, COHN, J. Am. Chem. Sot., 7 z ( | 9 5 o) 1471. R. B. I'IURLBERT, l-I. SCHbIITZ, A. 1'-'. BRUMM t~T V. R. I'OTTIIR, J . !1iol. Chem., 209 (t054) "3. e p . S. CHIIN, L. Y. TORIBARA liT H. WARNI';R, ,'|rod. Chem., .,8 (tO56) t756. ? D. KIilLIN l i t E. F, HAR'rRI~F., Biochem. J., 42 (1948) z3o. s j , H, ROlL J. Biol, Chem., , l z (t955) 335, 0 R. G. HANSE.~, R. A. I:RRF.LANO RT H. M. Sco'rT, J. Biol. Chem., 210 (1956) 391.
Re~u le 24 octobre, x96x Biochim. Biophys...h:/a, .57 (196z) 370-.381
Nature of the coenzyme binding site of bovine malate dehydrogenase from the displacement of the ultraviolet absorption maximum of DPNH in the presence of enzyme,, have obtained a value of o.8 5 mole of DPNH bound per mole of pig-heart mitochondrial malate dehydrogenase (L.malate: P F L E I D E R E R AND H O H N H O L Z 1,
Biochim. Biophys. :lclao .57 (I062) 3 8 1 - 3 8 3
382
SHORT COMMUNICATIONS
DPN+ o.,ddoreductase, EC I.I.L37), calculated on the basis of a molecular weight of 40 ceo. This displacement of the absorption ma.,dmum was abolished by organic compounds of mercury, thereby suggesting an interaction with sulfhydryl groups. The " current investigation reports the reaction of iodoacetamide with purified bovine-heart mitochondrial malate dehydrogenase mand some preliminary findings with regard to the enzyme structure. Bovine-heart mitochondrial malate dehydrogenase activity was completely abolished after 90 min in the presence of 5 mM iodoacetamide, buffered with Io mM sodium phosphate, pH 7.4. These findings are in agreement with those of GREENa, who found that pig-heart malate dehydrogenase was inhibited by 3o mM iodoacetate. BARRON AND SINGER4, on the other hand, found that pigeon-heart malate dehydrogenase activity was unaffected by 3 mM iodoacetamide, although #-chloromercuribenzoate was an effective inhibitor. Both the pig-heart and bovine-heart "mitochondrial" enzymes have been shown to be inhibited by p-chloromercuribenzoate while the bovine-heart "cytoplasmic" form was insensitive6, 6. Iodoacctamide (Io raM) was allowed to react with the purified bovine-heart enzyme (3° mg) while a pH of 8.5 was maintained by titration with base of the HI released (using a Radiometer Titrator TTTIa). Within about 9° rain the iodoacetamide had reacted, and a secondary reaction had set in, similar to that reported in the reaction of iodoacetate with creatine phosphokinase7. A blank run without the enzyme was made to correct for the titration of COmtaken up from the atmosphere. At the completion of the primary reaction the enzyme was inactive, and 4-5 moles of iodoacetamide had reacted per mole of enzyme {calculated on the basis of a molecular weight of 65 ooo). The presence of I mM DPNH did not prevent the reaction and inhibition of the enzyme by iodoacetamide, and the iodoacetamide-treated malate dehydrogenase (which had been dialyzed against water) bound DPNH one-fifth as well as native enzyme in equihbrium dialysis (Fig. I). The bound coenzyme on the native malate dehydrogenase could not be removed by exhaustive dialysis against water or dilute salt solutions, but wvs removed by an ethanol precipitation of the enzyme, followed by dialysis against buffer. The binding of the coenzyme to the enzyme decreased with an increase in pH. Iodoacetamide-treated malate dehydrogenase was dialyzed against water and lyophilized. A sample of the enzyme derivative was hydrolyzed in 5.7 N HCI for I8 h at Ixo ° and chromatographed on a Spinco Amino Acid Analyzer, Model I2o. Native enzyme was similarly treated. The analysis revealed that o.o2 pmole of S-carboxymethylcysteine was present per milligram of the iodoacctamide-treated malate dehydrogenase (corrected for 8-9 % decomposition of the derivative during hydrolysis as described in ref. 8). On the basis of a molecular weight of 65 ooo, this value gives I. 3 residues of cysteine carboxymethylated per mole of bovine-heart mitochondrial enzyme; this finding is in close agreement with the value of coenzyme bound per mole of pig-heart enzyme1. An amino acid analysis of an acid hydrolysate of the native enzyme showed 9-Io half-cystine residues present per mole of enzyme. Other workers have found a value of z2 for the same enzymea. These results are consistent with the view that a "free" sulfhydryl group on the protein reacts with the coenzyme during the enzymic reaction. The evidence here, however, does not represent rigorous proof of this hypothesis since data are lacking with regard to the effect of iodoacetamide on the tertiary structure of the protein, as well as the fact that not all moles of iodoacetBiochim. Biophys. Acta, 57 (I96~) 381-383
SHORT COMMUNICATIONS
383
a m i d e r e a c t i n g h a v e been z.ccountc~l for. A trace of e-c.~rboxymethyllysine was ~he only o t h e r d e r i v a t i v e detected. T h e present knowledge should now p e r m i t a n a p p r o a c h to the s t r u c t u r e of t h e " a c t i v e c e n t e r " of t h e e n z y m e b y t h e use of labelled iodoacetamide, followed b y proteinase digestion of t h e d e r i v a t i v e t h u s formed.
16
to
r
2. 0
tO
2.0
~.
~
3,0
4.0
N I#~'
Fig. L Equilibrium dialysis binding of D P N H to native and iodoacetamide-treated bovine-heart
mitochondrial malate dehydrngen,'me. Conditions: o.t M Tris-HCI, 0.05 M ED'FA, pH 8.1, 5 ~'. Dialysis was performed for -'4 h to insure equilibrium. The molar extinction coefficient of DPNH at 340 rap, 6.22, xos, was used in the calculation of the lfinding values. A, iodoacetamide-treated enzyme; B, native enzyme. O t h e r p r e l i m i n a r y s t r u c t u r a l work on the b o v i n e - h e a r t m i t o c h o n d r i a l e n z y m e h a s s h o w n t h e N - t e r m i n a l a m i n o acid to be alanine (by t h e fluorodinitrobenzene m e t h o d of Sanger). Carbox'ypeptidase ( D F P - t r e a t e d ) digestion of t h e n a t i v e e n z y m e yielded t h r e o n i n e , serine, a n d m e t h i o n i n e as t h e a m i n o acids released. Pig-heart m a l a t e d e h y d r o g e n a s e from C. F. Boehringer & Soenhe h a d t h e same N - t e r m i n a l g r o u p a n d released t h e same a m i n o acids in c a r b o x y p e p t i d a s e digestion. I n addition, b o t h t h e pig-heart a n d b o v i n e - h e a r t e n z y m e s c o n t a i n e d o - z mole of t r y p t o p h a n per mole of enzyme.
Biology Division, Oak Ridge National Laboratory*', Oak Ridge, Tenn. (U.S.A.)
F . C . GRIMM* D . G . DOHERTY
i G. PFLI~IDERERAND E. HOHNHOLZ, Bioehem. Z., 331 (1959) 245. tl F. C. GRIMMAND D. G. DOIIERTY, J. Biol. Chem., 23b (106I) 198o. s D. E. GREEN, Biochem. J., 3o (I936) 2005. 4 E. S. G, BARREN AND T. P. SING,;R, J. Biol. Chem., 157 (i945) 2zl. s R. G. WoLI~I~AND J. B. NIELANDS, J. Biol. Chem., 2~1 (1956) 61. a L. SIEOnL AND S. ENOLAnO, Federation Prec., zo (196t) 239. * D. C. WATTS, B. R. RAmN AND E. M. CROOK, Bioehim. Biophys..~cta, 48 (196t) 38o. s R. D, Cot.g, W. H. STr~:INAND S, MoonR, J. Biol. Chem., z33 (1958) I359. " Present address: The Rockefeller Institute, New York 2z, N.Y. (U.S.A.). "* Ol~era~ed by Union Carbide Corporation for the U.S. Atomic Energy Commission. R e c e i v e d O c t o b e r xzth, x96x
Biothim. Biophys. Acts, 57 (196z) 38x-383
38I.
accord avec la constatation fare par HANSEN. FREELAND ET SCOTT9 dans le foie de poulet soumis it un r6gime riche en galactose. Mais ces auteurs n'ont pas fourni de donn6es quantitatives concernant les deux UDP-hexoses. Les rfisultats du dosage que nous avons effectu6 (Tableau I) nous ont r6v~16 une r6duction de l'UDP-glucose g6n6ralement de l'ordre de 5o°/. et un accroissement de l'UDP-galactose du quintuple ou plus. Ce ph6nom~,ne nous indique clue dans le cristallin in vivo, la production de TABLEAU
II
TENF,UR I';N GLYCOGI~'NE I)l'~ CRISTALLINS DI~ RATS ~G~S D|t 7 SEMAINF.S SOUMIS 2~. UN R~GIME ~ BASE DIe GLUCOSE OU DE GAI.ACTOSb; PENDANT ~ JOURS
die,~gitl,,, Glucose Galactost~
Expt. t
Expt, ~
Expt. 3
4~ 2o
46 2!
3° t5
l'UDP-galactose h partir du galactose x-phosphate + UTP est plus rapide que l'6pim~risation de l'UDP-galactose donnant de l'UDP-glucose. I1 en r~sulte une accumulation d'UDP-galactose avec r&luction tie l'UDP-glucose chez les rats soumis/t un r~gime fiche en galactose. De plus la eompftition du galactose I-phosphate et du glucose x-phosphate pour la formation ties UDP-hexoses par l'uridyltransf6rase semble ~galement intervenir. On en trouve les r6percussions dans la baisse du taux de l'UDP-glucose et du glycogtXne du cristallin (Tableau II). Ainsi it c6t6 de l'accumulation du galactose x-phusphate signal6e par SCHWARZ~T GOLBERC;~ une autre perturbation m6tabolique cons6cutive it la surcharge du r6gime riche en galactose a pu ~tre d6cel~e. I1 serait 6.videmment int6ressant de rechercher le mt~me ph6nom~-ne dabs les galactost~.mies par "erreur m6tabolique" con#nitale. lnstitut de Chimie Biologique, Facuit~ de Mddecine, Strasbour~ (France)
J. KI.~Tm P. MANDEL
I p. MANDEL I~T J . KLI~THI, liiochim, ltiophys...tcta, z8 (I958) 199. I V. SCItWARZ ~T L. GOLBF.RO, ltiochim, ltiophys...hta, t8 (1055) 310. a L. MANDRL, M. Wt.~TZVmTH, M. JACOR, M. P~:aRV V:T P, MANDt.:L, Compt. trod. soc. biol., t51. (1957) 993. 4 W. E, COHN, J. Am. Chem. Sot., 7 z ( | 9 5 o) 1471. R. B. I'IURLBERT, l-I. SCHbIITZ, A. 1'-'. BRUMM t~T V. R. I'OTTIIR, J . !1iol. Chem., 209 (t054) "3. e p . S. CHIIN, L. Y. TORIBARA liT H. WARNI';R, ,'|rod. Chem., .,8 (tO56) t756. ? D. KIilLIN l i t E. F, HAR'rRI~F., Biochem. J., 42 (1948) z3o. s j , H, ROlL J. Biol, Chem., , l z (t955) 335, 0 R. G. HANSE.~, R. A. I:RRF.LANO RT H. M. Sco'rT, J. Biol. Chem., 210 (1956) 391.
Re~u le 24 octobre, x96x Biochim. Biophys...h:/a, .57 (196z) 370-.381
Nature of the coenzyme binding site of bovine malate dehydrogenase from the displacement of the ultraviolet absorption maximum of DPNH in the presence of enzyme,, have obtained a value of o.8 5 mole of DPNH bound per mole of pig-heart mitochondrial malate dehydrogenase (L.malate: P F L E I D E R E R AND H O H N H O L Z 1,
Biochim. Biophys. :lclao .57 (I062) 3 8 1 - 3 8 3
382
SHORT COMMUNICATIONS
DPN+ o.,ddoreductase, EC I.I.L37), calculated on the basis of a molecular weight of 40 ceo. This displacement of the absorption ma.,dmum was abolished by organic compounds of mercury, thereby suggesting an interaction with sulfhydryl groups. The " current investigation reports the reaction of iodoacetamide with purified bovine-heart mitochondrial malate dehydrogenase mand some preliminary findings with regard to the enzyme structure. Bovine-heart mitochondrial malate dehydrogenase activity was completely abolished after 90 min in the presence of 5 mM iodoacetamide, buffered with Io mM sodium phosphate, pH 7.4. These findings are in agreement with those of GREENa, who found that pig-heart malate dehydrogenase was inhibited by 3o mM iodoacetate. BARRON AND SINGER4, on the other hand, found that pigeon-heart malate dehydrogenase activity was unaffected by 3 mM iodoacetamide, although #-chloromercuribenzoate was an effective inhibitor. Both the pig-heart and bovine-heart "mitochondrial" enzymes have been shown to be inhibited by p-chloromercuribenzoate while the bovine-heart "cytoplasmic" form was insensitive6, 6. Iodoacctamide (Io raM) was allowed to react with the purified bovine-heart enzyme (3° mg) while a pH of 8.5 was maintained by titration with base of the HI released (using a Radiometer Titrator TTTIa). Within about 9° rain the iodoacetamide had reacted, and a secondary reaction had set in, similar to that reported in the reaction of iodoacetate with creatine phosphokinase7. A blank run without the enzyme was made to correct for the titration of COmtaken up from the atmosphere. At the completion of the primary reaction the enzyme was inactive, and 4-5 moles of iodoacetamide had reacted per mole of enzyme {calculated on the basis of a molecular weight of 65 ooo). The presence of I mM DPNH did not prevent the reaction and inhibition of the enzyme by iodoacetamide, and the iodoacetamide-treated malate dehydrogenase (which had been dialyzed against water) bound DPNH one-fifth as well as native enzyme in equihbrium dialysis (Fig. I). The bound coenzyme on the native malate dehydrogenase could not be removed by exhaustive dialysis against water or dilute salt solutions, but wvs removed by an ethanol precipitation of the enzyme, followed by dialysis against buffer. The binding of the coenzyme to the enzyme decreased with an increase in pH. Iodoacetamide-treated malate dehydrogenase was dialyzed against water and lyophilized. A sample of the enzyme derivative was hydrolyzed in 5.7 N HCI for I8 h at Ixo ° and chromatographed on a Spinco Amino Acid Analyzer, Model I2o. Native enzyme was similarly treated. The analysis revealed that o.o2 pmole of S-carboxymethylcysteine was present per milligram of the iodoacctamide-treated malate dehydrogenase (corrected for 8-9 % decomposition of the derivative during hydrolysis as described in ref. 8). On the basis of a molecular weight of 65 ooo, this value gives I. 3 residues of cysteine carboxymethylated per mole of bovine-heart mitochondrial enzyme; this finding is in close agreement with the value of coenzyme bound per mole of pig-heart enzyme1. An amino acid analysis of an acid hydrolysate of the native enzyme showed 9-Io half-cystine residues present per mole of enzyme. Other workers have found a value of z2 for the same enzymea. These results are consistent with the view that a "free" sulfhydryl group on the protein reacts with the coenzyme during the enzymic reaction. The evidence here, however, does not represent rigorous proof of this hypothesis since data are lacking with regard to the effect of iodoacetamide on the tertiary structure of the protein, as well as the fact that not all moles of iodoacetBiochim. Biophys. Acta, 57 (I96~) 381-383
SHORT COMMUNICATIONS
383
a m i d e r e a c t i n g h a v e been z.ccountc~l for. A trace of e-c.~rboxymethyllysine was ~he only o t h e r d e r i v a t i v e detected. T h e present knowledge should now p e r m i t a n a p p r o a c h to the s t r u c t u r e of t h e " a c t i v e c e n t e r " of t h e e n z y m e b y t h e use of labelled iodoacetamide, followed b y proteinase digestion of t h e d e r i v a t i v e t h u s formed.
16
to
r
2. 0
tO
2.0
~.
~
3,0
4.0
N I#~'
Fig. L Equilibrium dialysis binding of D P N H to native and iodoacetamide-treated bovine-heart
mitochondrial malate dehydrngen,'me. Conditions: o.t M Tris-HCI, 0.05 M ED'FA, pH 8.1, 5 ~'. Dialysis was performed for -'4 h to insure equilibrium. The molar extinction coefficient of DPNH at 340 rap, 6.22, xos, was used in the calculation of the lfinding values. A, iodoacetamide-treated enzyme; B, native enzyme. O t h e r p r e l i m i n a r y s t r u c t u r a l work on the b o v i n e - h e a r t m i t o c h o n d r i a l e n z y m e h a s s h o w n t h e N - t e r m i n a l a m i n o acid to be alanine (by t h e fluorodinitrobenzene m e t h o d of Sanger). Carbox'ypeptidase ( D F P - t r e a t e d ) digestion of t h e n a t i v e e n z y m e yielded t h r e o n i n e , serine, a n d m e t h i o n i n e as t h e a m i n o acids released. Pig-heart m a l a t e d e h y d r o g e n a s e from C. F. Boehringer & Soenhe h a d t h e same N - t e r m i n a l g r o u p a n d released t h e same a m i n o acids in c a r b o x y p e p t i d a s e digestion. I n addition, b o t h t h e pig-heart a n d b o v i n e - h e a r t e n z y m e s c o n t a i n e d o - z mole of t r y p t o p h a n per mole of enzyme.
Biology Division, Oak Ridge National Laboratory*', Oak Ridge, Tenn. (U.S.A.)
F . C . GRIMM* D . G . DOHERTY
i G. PFLI~IDERERAND E. HOHNHOLZ, Bioehem. Z., 331 (1959) 245. tl F. C. GRIMMAND D. G. DOIIERTY, J. Biol. Chem., 23b (106I) 198o. s D. E. GREEN, Biochem. J., 3o (I936) 2005. 4 E. S. G, BARREN AND T. P. SING,;R, J. Biol. Chem., 157 (i945) 2zl. s R. G. WoLI~I~AND J. B. NIELANDS, J. Biol. Chem., 2~1 (1956) 61. a L. SIEOnL AND S. ENOLAnO, Federation Prec., zo (196t) 239. * D. C. WATTS, B. R. RAmN AND E. M. CROOK, Bioehim. Biophys..~cta, 48 (196t) 38o. s R. D, Cot.g, W. H. STr~:INAND S, MoonR, J. Biol. Chem., z33 (1958) I359. " Present address: The Rockefeller Institute, New York 2z, N.Y. (U.S.A.). "* Ol~era~ed by Union Carbide Corporation for the U.S. Atomic Energy Commission. R e c e i v e d O c t o b e r xzth, x96x
Biothim. Biophys. Acts, 57 (196z) 38x-383