- Email: [email protected]
[45] Methoxycarbonyl-CoA disulfide: An active site=directed sulfhydryl reagent
584
SUBSTRATES, A N A L O G S , A N D I N H I B I T O R S
[45] M e t h o x y c a r b o n y l - C o A
[45]
Disulfide: An Active Site-Directed
Sulfhydryl Reagent j By Louis B. HERSH and DANIEL J. SMITH
The relative ease with which protein sulfhydryl groups react with a variety of compounds has lead to the preparation of a large number of sulfhydryl reagents. Kenyon and Bruice la have classified these reagents according to the groups that are delivered: blocking and labeling groups, reporter groups, cross-linking groups, and afffinity-labeling groups. Recently Smith and Kenyon 2 and Smith et al.3 used methoxyO
II
carbonyl-alkyl disulfides (CH30---C--S--SR) as highly reactive sulfhydryl group blocking reagents. The description of these reagents led us to the design and synthesis of a similar reagent that maintains the high reactivity of methoxycarbonyl alkyl disulfides toward sulfhydryl groups and in addition can act as an affinity labeling reagent for enzymes using acyl-CoA as substrate. Methoxycarbonyl-CoA disulfide, O
II
C H 3 0 - - C - - S - - S C o A has been shown to exhibit selectivity for a sulfhydryl group in the acetyl-CoA binding site of the enzyme choline acetyltransferase. Preparation of Methoxycarbonyl-CoA Disulfide Methoxycarbonyl-CoA disulfide is prepared by reaction of free CoA with methoxycarbonylsulfenyi chloride. The latter compound is prepared in a two-step synthesis starting with trichloromethanesulfenyl chloride. 4
i This work was s u p p o r t e d in part by Grant A M 13443 from T h e National Institutes of H e a l t h , Grant 1-391 from the Robert A. Welch Foundation, and Grant 10850-G4 from The Petroleum R e s e a r c h Foundation. la G. K e n y o n and T. C. Bruice, this series, Vol. 47, p. 407. 2 D. J. Smith and G. L. K e n y o n , J. Biol. Chem. 249, 3317 (1974). 3 D. J. Smith, E. T. Maggio, and G. L. Kenyon, Biochemistry 14, 766 (1975). 4 G. Z u m a c h and E. Kuhle, Angew Chem., Int. Ed. Engl. 9, 54 (1970).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
[45]
METHOXYCARBON Y L - C o A DISULFIDE
(1) C l a C - S - C l
H~S04 H20
0 II ~,- c 1 - c - s c 1
O
585
~ 2HC1 O
II
(2) C1--C--SC1 + CH3OH
tl
~ CHaO-C
O
II (3) CHaO--C--SCI ~ CoASH
SC1 + HC1 O
II
~" CH30--C--S--SCoA + HC1
Preparation of Methoxycarbonylsulfenyl Chloride 4 Trichloromethanesulfenyl chloride (1 mol, 186 g) is added to 211 g of concentrated sulfuric acid and 18 g of water. The mixture is stirred at 45-50 ° in a well-vented hood until hydrogen chloride evolution ceases (approximately 5 hr). After cooling to room temperature, two layers are formed. The top brown colored layer is separated and distilled (98° at 760 mm) to yield 110 g (85% yield) of the yellow chlorocarbonylsulfenyl chloride. Care should be exercised in handling this toxic compound. To 52 g of chlorocarbonylsulfenyl chloride in a round-bottom flask is slowly added 14 g of anhydrous methanol. A reflux condenser is attached to the round-bottom flask, and the mixture is stirred at 55 ° for 3 hr. Thirty-five grams (69% yield) of methoxycarbonylsulfenyl chloride are obtained by vacuum distillation at 73-74 ° and 100 mm. The methoxycarbonylsulfenyl chloride can be stored in a freezer in a well-stoppered flask, but should be used as soon as possible.
Preparation of MethoxycarbonyI-CoA Disulfide 5 To a chilled solution of 0.06 mmol ( - 5 0 mg) of CoA, lithium salt in 2 ml of anhydrous methanol, is added 0.32 mmol (40 rag) of freshly distilled methoxycarbonylsulfenyl chloride. The reaction mixture is gently stirred for 3 hr at 3-5 ° (ice bath); after this time the product is precipitated by the addition of anhydrous ether. The white solid is collected by filtration and washed several times with ether. The product is taken up in a minimal volume of water and lyophilized. At this stage in the synthesis the product is contaminated with a small amount of oxidized CoA. In order to further purify the product, it is dissolved in a minimal volume of 3 mM HC1 and chromatographed on a column of DEAE-cellulose (2 × 10 cm) previously equilibrated with 3 mM HCI. Elution of methyoxycarbonyl-CoA disulfide is accomplished with a linear LiC1 gradient established between 750 ml of L. B. Hersh, R. V. Nair, and D. J. S m i t h , J . Biol. Chem. 254, 11988 (1979).
586
SUBSTRATES, ANALOGS, AND INHIB1TORS
[45]
3 m M HC1 and 750 ml of 0.3 M LiC1 in 3 m M HC1. M e t h o x y c a r b o n y l - C o A disulfide, identified by its a b s o r b a n c e at 260 nm, elutes between 0.0650.085 M LiC1. The column fractions containing m e t h o x y c a r b o n y l - C o A disulfide are lyophilized, and the resulting white solid is extracted several times with m e t h a n o l - a c e t o n e (1 : 10, v/v) to r e m o v e m o s t of the LiCI. The final product which is obtained in 70-80% yield, is dried under v a c u u m and stored desiccated in a refrigerator. Comment
The methyl group of m e t h o x y c a r b o n y l - C o A disulfide can be replaced with a variety of alkyl groups by substituting different alcohols for methanol in the reaction with chlorocarbonylsulfenyl chloride.4 Radiolabeled reagent can be p r e p a r e d by using all-labeled CoA. Characterization of M e t h o x y c a r b o n y l - C o A Disulfide The ultraviolet s p e c t r u m of m e t h o x y c a r b o n y l - C o A disulfide at p H 7 is identical to that o f free CoA, and therefore the concentration of an aqueous solution can be m e a s u r e d using Emit of 14.2 at 260 nm. The N M R s p e c t r u m o f the c o m p o u n d is also the same as that o f free C o A with the exception that a single new p e a k at 3.78 6 is observed. The chemical shift and integration of this additional p e a k is consistent with that e x p e c t e d for the three additional protons o f the m e t h o x y group. M e t h o x y c a r b o n y l - C o A disulfide migrates as a single ultraviolet compound, R f 0.85, on W h a t m a n No. I p a p e r using the solvent s y s t e m isobutyric a c i d - c o n c e n t r a t e d N H 4 O H - w a t e r (54 : 4 : 39, v/v/v). Treatment with 20 m M dithiothreitol for 30 rain at r o o m t e m p e r a t u r e quantitatively liberates CoA. Reaction of Methoxycarbonyl-CoA Disulfide with Choline Acetyltransferase Choline acetyltransferase catalyzes the reversible formation o f acetylcholine from a c e t y l - C o A and choline. A c e t y l - C o A + choline ~,~ acetylcholine + C o A A n u m b e r o f studies have shown that this e n z y m e is sensitive to a variety o f sulfhydryl reagents, 6 - ' ' although the n u m b e r and localization of 6 B. Mannervick and B. Sorbo, Biochem. Pharmacol. 19, 2509 (1970). 7 R. Berman-Resiberg, Biochim. Biophys. Acta 14, 442 (1954). J. Schuberth, Biochim. Biophys. Acla 122, 470 (1966). R. Roskoski, Jr., J. Biol. Chem. 249, 2156 (1974). ,o R. Roskoski, Jr., C. T. Lim, and L. M. Rosbokoski, Biochemistry 14, 5105 (1975). " S. F. Currier and H. G. Mautner, Biochemistry 16, 1944 (1977).
[46]
o J - B R O M O A C E T A M I D O FAXTY ACIDS
587
reactive sulfhydryl groups has not been determined. MethoxycarbonylCoA disulfide reacts with a sulfhydryl group in the acetyl-CoA binding site of the e n z y m e 35-fold faster than DTNB.5 Substitution of the CoA moiety with small alkyl group virtually abolished reaction at the acetylCoA site. M e t h o x y c a r b o n y l - C o A disulfide reacts - 3 5 times more slowly than D T N B with a second, more exposed sulfhydryl group on the enzyme. These results demonstrate that the sulfhydryl reagent m e t h o x y c a r b o n y l - C o A disulfide is an active-site-directed acyl-CoA analog.
[46] A f f i n i t y L a b e l i n g
with oJ-Bromoacetamido
Fatty
Acids
and Analogs By BRYCE V. PLAPP
and
WEN-SHERNG
CHEN
Using a knowledge of the three-dimensional structure of horse liver alcohol dehydrogenase, we designed some co-bromoacetamido fatty acids t to bind to the e n z y m e - N A D + complex and alkylate the sulfur of methionine residue 306, which forms part of the substrate binding pocket. 2 F a t t y acids were chosen as the affinity moieties since they bind tightly to the e n z y m e • N A D ÷ complex and competitively inhibit binding of ethanol .3 Alternatively, fatty acid amides could be used since they bind to the e n z y m e • N A D H complex. It is assumed that the carboxylate or c a r b o x a m i d e groups bind to the zinc in the active site o f the e n z y m e c o e n z y m e complexes. The lengths and shapes of the b r o m a c e t a m i d o reagents were varied in order to obtain c o m p o u n d s that would bridge the 14 ]k between the zinc and Met-306. In the absence of N A D ÷, we expected that the reagents could bind in alternative w a y s - - f o r instance, with the carboxylate interacting with the anion binding site furnished by Arg-47 and L y s - 2 2 8 - - a n d react with cysteine residues 46 or 174, as in the inactivation by iodoacetate. 4 Reagents of just the right size and shape appear to react as predicted, and we n a m e d them " a m b i v a l e n t active-site-directed r e a g e n t s " b e c a u s e they can react in two different ways with the enzyme; their ambivalence is resolved by coenzyme. 1 W.-S. Chen and B. V. Plapp, Biochemistry 17, 4916 (1978). 2 C.-I. Br~indfn, H. J/Srnvall, H. Eklund, and B. Furugren, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. I 1, p. 104. Academic Press, New York, 1975. 3 A. D. Winer and H. Theorell, Acta Chem. Scand. 14, 1729 (1960). 4 E. Zeppezauer, H. J/~rnvall, and I. Ohlsson, Eur. J. Biochem. 58, 95 (1975).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright (~ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181972-8
SUBSTRATES, A N A L O G S , A N D I N H I B I T O R S
[45] M e t h o x y c a r b o n y l - C o A
[45]
Disulfide: An Active Site-Directed
Sulfhydryl Reagent j By Louis B. HERSH and DANIEL J. SMITH
The relative ease with which protein sulfhydryl groups react with a variety of compounds has lead to the preparation of a large number of sulfhydryl reagents. Kenyon and Bruice la have classified these reagents according to the groups that are delivered: blocking and labeling groups, reporter groups, cross-linking groups, and afffinity-labeling groups. Recently Smith and Kenyon 2 and Smith et al.3 used methoxyO
II
carbonyl-alkyl disulfides (CH30---C--S--SR) as highly reactive sulfhydryl group blocking reagents. The description of these reagents led us to the design and synthesis of a similar reagent that maintains the high reactivity of methoxycarbonyl alkyl disulfides toward sulfhydryl groups and in addition can act as an affinity labeling reagent for enzymes using acyl-CoA as substrate. Methoxycarbonyl-CoA disulfide, O
II
C H 3 0 - - C - - S - - S C o A has been shown to exhibit selectivity for a sulfhydryl group in the acetyl-CoA binding site of the enzyme choline acetyltransferase. Preparation of Methoxycarbonyl-CoA Disulfide Methoxycarbonyl-CoA disulfide is prepared by reaction of free CoA with methoxycarbonylsulfenyi chloride. The latter compound is prepared in a two-step synthesis starting with trichloromethanesulfenyl chloride. 4
i This work was s u p p o r t e d in part by Grant A M 13443 from T h e National Institutes of H e a l t h , Grant 1-391 from the Robert A. Welch Foundation, and Grant 10850-G4 from The Petroleum R e s e a r c h Foundation. la G. K e n y o n and T. C. Bruice, this series, Vol. 47, p. 407. 2 D. J. Smith and G. L. K e n y o n , J. Biol. Chem. 249, 3317 (1974). 3 D. J. Smith, E. T. Maggio, and G. L. Kenyon, Biochemistry 14, 766 (1975). 4 G. Z u m a c h and E. Kuhle, Angew Chem., Int. Ed. Engl. 9, 54 (1970).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
[45]
METHOXYCARBON Y L - C o A DISULFIDE
(1) C l a C - S - C l
H~S04 H20
0 II ~,- c 1 - c - s c 1
O
585
~ 2HC1 O
II
(2) C1--C--SC1 + CH3OH
tl
~ CHaO-C
O
II (3) CHaO--C--SCI ~ CoASH
SC1 + HC1 O
II
~" CH30--C--S--SCoA + HC1
Preparation of Methoxycarbonylsulfenyl Chloride 4 Trichloromethanesulfenyl chloride (1 mol, 186 g) is added to 211 g of concentrated sulfuric acid and 18 g of water. The mixture is stirred at 45-50 ° in a well-vented hood until hydrogen chloride evolution ceases (approximately 5 hr). After cooling to room temperature, two layers are formed. The top brown colored layer is separated and distilled (98° at 760 mm) to yield 110 g (85% yield) of the yellow chlorocarbonylsulfenyl chloride. Care should be exercised in handling this toxic compound. To 52 g of chlorocarbonylsulfenyl chloride in a round-bottom flask is slowly added 14 g of anhydrous methanol. A reflux condenser is attached to the round-bottom flask, and the mixture is stirred at 55 ° for 3 hr. Thirty-five grams (69% yield) of methoxycarbonylsulfenyl chloride are obtained by vacuum distillation at 73-74 ° and 100 mm. The methoxycarbonylsulfenyl chloride can be stored in a freezer in a well-stoppered flask, but should be used as soon as possible.
Preparation of MethoxycarbonyI-CoA Disulfide 5 To a chilled solution of 0.06 mmol ( - 5 0 mg) of CoA, lithium salt in 2 ml of anhydrous methanol, is added 0.32 mmol (40 rag) of freshly distilled methoxycarbonylsulfenyl chloride. The reaction mixture is gently stirred for 3 hr at 3-5 ° (ice bath); after this time the product is precipitated by the addition of anhydrous ether. The white solid is collected by filtration and washed several times with ether. The product is taken up in a minimal volume of water and lyophilized. At this stage in the synthesis the product is contaminated with a small amount of oxidized CoA. In order to further purify the product, it is dissolved in a minimal volume of 3 mM HC1 and chromatographed on a column of DEAE-cellulose (2 × 10 cm) previously equilibrated with 3 mM HCI. Elution of methyoxycarbonyl-CoA disulfide is accomplished with a linear LiC1 gradient established between 750 ml of L. B. Hersh, R. V. Nair, and D. J. S m i t h , J . Biol. Chem. 254, 11988 (1979).
586
SUBSTRATES, ANALOGS, AND INHIB1TORS
[45]
3 m M HC1 and 750 ml of 0.3 M LiC1 in 3 m M HC1. M e t h o x y c a r b o n y l - C o A disulfide, identified by its a b s o r b a n c e at 260 nm, elutes between 0.0650.085 M LiC1. The column fractions containing m e t h o x y c a r b o n y l - C o A disulfide are lyophilized, and the resulting white solid is extracted several times with m e t h a n o l - a c e t o n e (1 : 10, v/v) to r e m o v e m o s t of the LiCI. The final product which is obtained in 70-80% yield, is dried under v a c u u m and stored desiccated in a refrigerator. Comment
The methyl group of m e t h o x y c a r b o n y l - C o A disulfide can be replaced with a variety of alkyl groups by substituting different alcohols for methanol in the reaction with chlorocarbonylsulfenyl chloride.4 Radiolabeled reagent can be p r e p a r e d by using all-labeled CoA. Characterization of M e t h o x y c a r b o n y l - C o A Disulfide The ultraviolet s p e c t r u m of m e t h o x y c a r b o n y l - C o A disulfide at p H 7 is identical to that o f free CoA, and therefore the concentration of an aqueous solution can be m e a s u r e d using Emit of 14.2 at 260 nm. The N M R s p e c t r u m o f the c o m p o u n d is also the same as that o f free C o A with the exception that a single new p e a k at 3.78 6 is observed. The chemical shift and integration of this additional p e a k is consistent with that e x p e c t e d for the three additional protons o f the m e t h o x y group. M e t h o x y c a r b o n y l - C o A disulfide migrates as a single ultraviolet compound, R f 0.85, on W h a t m a n No. I p a p e r using the solvent s y s t e m isobutyric a c i d - c o n c e n t r a t e d N H 4 O H - w a t e r (54 : 4 : 39, v/v/v). Treatment with 20 m M dithiothreitol for 30 rain at r o o m t e m p e r a t u r e quantitatively liberates CoA. Reaction of Methoxycarbonyl-CoA Disulfide with Choline Acetyltransferase Choline acetyltransferase catalyzes the reversible formation o f acetylcholine from a c e t y l - C o A and choline. A c e t y l - C o A + choline ~,~ acetylcholine + C o A A n u m b e r o f studies have shown that this e n z y m e is sensitive to a variety o f sulfhydryl reagents, 6 - ' ' although the n u m b e r and localization of 6 B. Mannervick and B. Sorbo, Biochem. Pharmacol. 19, 2509 (1970). 7 R. Berman-Resiberg, Biochim. Biophys. Acta 14, 442 (1954). J. Schuberth, Biochim. Biophys. Acla 122, 470 (1966). R. Roskoski, Jr., J. Biol. Chem. 249, 2156 (1974). ,o R. Roskoski, Jr., C. T. Lim, and L. M. Rosbokoski, Biochemistry 14, 5105 (1975). " S. F. Currier and H. G. Mautner, Biochemistry 16, 1944 (1977).
[46]
o J - B R O M O A C E T A M I D O FAXTY ACIDS
587
reactive sulfhydryl groups has not been determined. MethoxycarbonylCoA disulfide reacts with a sulfhydryl group in the acetyl-CoA binding site of the e n z y m e 35-fold faster than DTNB.5 Substitution of the CoA moiety with small alkyl group virtually abolished reaction at the acetylCoA site. M e t h o x y c a r b o n y l - C o A disulfide reacts - 3 5 times more slowly than D T N B with a second, more exposed sulfhydryl group on the enzyme. These results demonstrate that the sulfhydryl reagent m e t h o x y c a r b o n y l - C o A disulfide is an active-site-directed acyl-CoA analog.
[46] A f f i n i t y L a b e l i n g
with oJ-Bromoacetamido
Fatty
Acids
and Analogs By BRYCE V. PLAPP
and
WEN-SHERNG
CHEN
Using a knowledge of the three-dimensional structure of horse liver alcohol dehydrogenase, we designed some co-bromoacetamido fatty acids t to bind to the e n z y m e - N A D + complex and alkylate the sulfur of methionine residue 306, which forms part of the substrate binding pocket. 2 F a t t y acids were chosen as the affinity moieties since they bind tightly to the e n z y m e • N A D ÷ complex and competitively inhibit binding of ethanol .3 Alternatively, fatty acid amides could be used since they bind to the e n z y m e • N A D H complex. It is assumed that the carboxylate or c a r b o x a m i d e groups bind to the zinc in the active site o f the e n z y m e c o e n z y m e complexes. The lengths and shapes of the b r o m a c e t a m i d o reagents were varied in order to obtain c o m p o u n d s that would bridge the 14 ]k between the zinc and Met-306. In the absence of N A D ÷, we expected that the reagents could bind in alternative w a y s - - f o r instance, with the carboxylate interacting with the anion binding site furnished by Arg-47 and L y s - 2 2 8 - - a n d react with cysteine residues 46 or 174, as in the inactivation by iodoacetate. 4 Reagents of just the right size and shape appear to react as predicted, and we n a m e d them " a m b i v a l e n t active-site-directed r e a g e n t s " b e c a u s e they can react in two different ways with the enzyme; their ambivalence is resolved by coenzyme. 1 W.-S. Chen and B. V. Plapp, Biochemistry 17, 4916 (1978). 2 C.-I. Br~indfn, H. J/Srnvall, H. Eklund, and B. Furugren, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. I 1, p. 104. Academic Press, New York, 1975. 3 A. D. Winer and H. Theorell, Acta Chem. Scand. 14, 1729 (1960). 4 E. Zeppezauer, H. J/~rnvall, and I. Ohlsson, Eur. J. Biochem. 58, 95 (1975).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright (~ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181972-8