Biochemistry of Lipoic Acid

Biochemistry of Lipoic Acid LESTER ,J. REED Clayton Foundation Biocheniical Institute und the Department of Chamisiry, The University of Texas, A u s t i n , Texas Page I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Chemistry of Lipoic Acid.. . . . . . . . . . ................................ A. Oxidation and Reduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ring Strain.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biological Function of Lipoic Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. I n Oxidative Decarboxylation of a-Keto Acids.. . . . . . . . . . . . . . . . . . . . . . . B. Other Possible Roles of Lipoic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2 4 4 4 31 34 34

I. INTRODUCTION Lipoic acid was discovered independently in several laboratories in the late 1940’s as a growth factor and requirement for pyruvate oxidation for certain microorganisms. The trivial names “acetate-replacing factor” (Guirard et al., 1946), ‘(pyruvate oxidation factor” (O’Kane and Gunsalus, 1948), “protogen” (Kidder and Dewey, 1949; Stokstad et al., 1949), and “B. R. factor” (Kline and Barker, 1950) were used to designate the biologically active substance prior to its isolation and identification. When a crystalline organic acid which was highly active in the acetate-replacing factor and pyruvate oxidation factor assays was isolated by Reed et al. (1951a) from the water-insoluble residue of beef liver, the trivial name “a-lipoic acid” was proposed to designate this specific substance. The prefix a- was not used in a chemical sense, but to distinguish the isolated compound from structurally related substances in extracts of biological materials. Subsequently, Patterson et al. (1951) isolated an oxidation product (sulfoxide) of a-lipoic acid. When the structure of the former substance was established, Bullock et al. (1952) proposed the name “6thioctic acid” for the parent substance (a-lipoic acid). The American Society of Biological Chemists has recognized priority of the name “lipoic acid” and has adopted it as the trivial designation of 1,2-dithiolane-3valeric acid (I). It is interesting to note that within a period of about six years following 1

2 the discovery of lipoic acid, this substancc, was isolated and characterized and the basic features of its site and mode of action were established. ThiR H,?’

s.--

H C2 ‘CH(CH,),CO,H

I

s Lipoic acid (1)

represents an imprccedented rate of devclopment of knowledge of the nature arid metabolic role of s biocatalyst. The historical aspects of this development have been summarized elsewhere (Reed, 1057). Lipoic acid has been shown to be widely distributed among microorgsnisms, plants, arid animals. Most nutritional investigations with higher sriiInds have failed to show a. growth response to addcd lipoic acid. However, there is 110 doubt that this suhstancc plays a vital role in anirnal metabolism. Thus far the only well-defined role of lipoic acid is that of a. prosthetic group in multicmyme c~omplexcswhich c.tlt,zlym an oxidative decnrhoxylation of pyruvatc and u-kctoglutaratc to producc acetyl cocnxyiric A (CoA) and succinyl CoA, respectively, and reduced diphosphopyridine nucleotide (IIPNH). Considerable data have accumulated on the nature of these cnzymc complexes and the role of lipoic acid ill the sequence of reactions leading from thc a-keto acid to ncyl CoA arid 1)PNH. The object of the present article is critically to evaluate and organize these data with a view to presenting an integrated picture in terms of reaction mechanisms of lipoic acid function in a-keto acid oxidation. Brief consideration will be givcri to those aspects of the chemistry of lipoic acid which are deemed pertinent to its biological role.

11. CHEMISTRY OF LIPOICACID A. OXIDATION A N D REDUCTION During the course of work on the isolation of lipoic acid, the tendency of this substance to undergo oxidation to a sulfoxide ((3-lipoic acid) in the presence of oxygen or peroxidcs was noted (Reed et al., 1953b; Patterson et al., 1954). It appears that the sulfur atom a t C-8 of the carbon skeleton is in the oxidized state (11) (Rccd, 1960). Calvin and co-workers (Barltrop et al., 1954; Calvin, 1954) have speculated that (3-lipoic acid is a possible intermediate in the photosynthetic production of oxygen. However, there is no experimental evidence a t present for a biochemical role of this substance. Lipoic acid is oxidized by performic acid to 6,S-disulfooc.tanoic acid

3

BIOCHEMISTRY OF LIPOIC ACID

(Nawa et al., 1960) [Eq. (l)]. This reaction played an important role in elucidation of the nature of the functional form of lipoic acid (see Section 111, A, 5).

fi(

CHZ)4C02H

s-s 1

0

(3-Lipoic acid

(11 1

Lipnir acid is rrdwiblr at the dropping mercury electrode (Reed et al., 1953s; Ke, 1957). Thc half-wave potential a t pH 7.0 is -0.567 volt versus the saturated (dome1 electrode (Kr, 1957). This value corresponds to a rednction potential of -0.325 volt with respect to the standard hydrogen electrode. The rrduction potential of the dihydrolipoir acid-lipoic acid system has been calculated from the cquilibrium constant of the dihydroHC0,H

fi(CH2)4C02H

s--s

-

~ ( C H , ) , C 0 2 H HOSS

(1)

SOSH

lipoic dehydrogenase-catalyzed interaction with the DPNH-DPN system (Sanadi et al., 1959a; Massey, 1960a; Goldman, 1959). The value obtained at pH 7.1 and 22°C is -0.294 volt (Sanadi et al., 1959a), which is in fair agreement with the polarographic measurements. Lipoie acid is reduced easily to dihydrolipoic acid (6,8-dithioloctanoic acid) (111) by sodium borohydride (Wagner et al., 1956; Gunsalus et al., 1956). Cleavage of the dithiolane ring by thiols (thiol-disulfide interchange reaction) has hccn noted (Ilecd et al., 1953b; Rarltrop et al., 1954; Fava et al., 1957).

(~(cH,)~co,H HS

SH Dihydrolipoic acid

(III) Dihydrolipoic acid is readily oxidized to the disulfide (I) by a variety of reagents such as iodine (Bullock et al., 1954), oxygen in the presence of metal cations (Bullock et al., 19,54), ferricyanide (Hager, IS.%), and oiodosohenzoate (llciss, 1958).

4

LESTER J. REED

B. RINGSTRAIN Lipoic acid exhibits an absorption maximum a t 330 mp (e = 150), whereas that of linear disulfides is located a t 250 mg (Calvin, 1954; Barltrop el al., 1954). A survey of five-, six-, arid sevcn-membered ryclic disulfides revealed that the ahsorption rrinximiini is progressively displaced toward longer wavelengths as the size of the ring is decreased. This spectral shift was attributed by Calvin arid co-workers to an increase in ring strain. The unique reactivit,y and instability of 1 2-dithiolane pointed up this possibility. In view of thc suggestion of Calvin and co-workers that lipoic acid participatcs in the primary quantum conversion reaction of photosynthesis, attempts havc been made to determine the strain energy in the 1,Z-dithiolane ring. A ncccwary condition for Calvin’s hypothesis appeared to be that the strain arriount to 25-30 kcal/mole. Conformat iond analysis ir-idiwtcd a ring strain of 16-30 kcal/molc (Bcrgson and Schotte, 1958; )

Rarltrop ~t al., 1954). Ilow~vcr,experimental determinations of the strain energy in 1’2-dithiolanc and lipoic acid have given much smaller values. Barltrop ct 01. (1954) obtained a valur of -6.3 kcallmole from equilibrium measurcIwnts (as a funcation of temperature) of the reaction betwecn 1,2-dithiolanc and benzyl mercaptan [Eq. (2)]. It is pertinent to note that thc free ciiergy ( A F ) of the latter reaction is only -1.67 kcal/mole (at 24.1OC). Fava et a/. (1957) extended this study and obtained values of 5.3 arid 4.2 kcal/molc from equililxium measiiremcnt,s of the reaction between 1,2-dithiolanc and n-butane thiol and methyl thioglycolate, respectively. Suriner (1955) reported values of 4 kcal/mole for the strain energy in 1 ,2-dithioltznc and 3.5 kcal/mole for lipoic acid, based on mcasurement of the. heats of oxidation of the corresponding dithiols. It appears from these rxpmimcnt a1 results that thr fivr-membered ring as found in lipoic acid docs riot tshibit3undue strain. 111. I 3 1 o r , o a i c * . \ ~E’~JN(’TIO~IUE’ L r ~ o i chr~n

A.

I N ( ) X l r ) 4 ~ I V h ; I)E(’AliHOXYLATION

01 a-I
Lipoic acid participates in the coenzyme A (CoA)- and diphosphopyridine nucleotidc (DI”)-linkcd oxidative decarboxylation of a-keto acids [E:y. (3)]. There are alternate pathways of a-keto acid oxidation which do RCOC02H

+ COA-SH + IIPN

C)

RCO-S-COA

+ Con + DPNH + €I+

(3)

not involve lipoic acid ( s e ~Gunsalus, I854a, b, for a suninlary of these reactions). Evidcnce for participatioii of lipoic acid in reaction (3) is

BIOCHEMISTRY O F LIPOIC ACID

5

summarized below. First, lipoic acid is essential for oxidation of pyruvate, a-ketobutyrate, P-methyl-a-ketobutyrate, and 0-methyl-a-ketovalerate by lipoic acid-deficient Streptococcus faecalis cells (O’Kane and Gunsalus, 1948; Gunsalus, 1954b). Both the aerobic oxidation and the dismutation of 2 pyruvate

+

0 2 -+

2 pyruvate

+

+ 2 COZ acetate + COZ + lactate 2 acetate

(4) (5)

pyruvate, Eqs. (4) and (5), are lipoic acid dependent (O’Kane and Gunsalus, 1948), but not the nonoxidative decarboxylation leading to acetoin formation [Eq. (6)] (O’Kane, 1950; D o h and Gunsalus, 1951). Second, 2 pyruvate -+ acetoin

+ 2 COZ

(6 )

enzyme systems which catalyze reaction (3) contain significant amounts of “bound” lipoic acid and are arsenite sensitive (Gunsalus, 195413; Sariadi et al., 1952, 1959a, b ; Schweet and Cheslock, 1952; Koike et al., 1960a; Massey, 1960b). In agreement with the earlier results of Peters and coworkers (Peters et al., 1946; Stockcn and Thompson, 1946a, b) with the brain pyruvate oxidation system, the inhibition by arsenite (at to 10-4 M ) is reversed by dithiols, but not by monothiols, a result suggesting the presence of an essential dithiol st,ructure in these enzyme syst,ems. Third, release of bound lipoic acid from the Escherichia coli pyruvate and a-ketoglutarate dehydrogenation complexes by incubation with a hydrolytic enzyme, “lipoyl-X hydrolase,” results in a loss of ability to catalyze reaction ( 3 ) (Reed et al., 195813; Koike and Reed, 1960). Reincorporation of lipoic acid into the inactive preparations by incubation with lipoic acid, adenosine triphosphate (ATP), and a lipoic acid-activating enzyme system restores this activity. These inactivation and reactivation experiments provide direct evidence of lipoic acid involvement in reaction (3). The available evidence indicates that the oxidative decarboxy1at)ion of pyruvate and a-ketoglutarate represenkd by Eg. (3) proceeds via the sequence shown in Fig. 1. This scheme assigns to the bound lipoic acid acyl-generation, acyl-transfer, and electron-transfer functions (Reed, 1953; Gunsalus, 1954a). Enzyme systems which catalyze reaction (3) have been isolat,ed from mammalian and bacterial cells as organized units (multienzyme complexes) of high molecular weight. Thus the pyruvate dehydrogenation system of pigeon breast muscle (Schweet et al., 1952) and the a-ketjoglutarate dehydrogenation system of pig heart muscle (Sanadi et al., 1952) were isolated as units with molecular weights of approximately 4 million and 2 million, respectively. Pyruvate and a-kct)ogIut,arate dehydrogenation systems have been isolated from 17.coli as complexes with molecular weights of 4.8 and 2.4 million, respectively (Koike et aE., 1960a). The E. coli pyruvate dehydrogenation complex has been resolved into

LESTER J. REED

0 II RCC0,H -!-

[RCHO-TPP]-E,

coz

TPP-E,

(8)

0 ~ ( C H d , C -II- E ,

(!I)

HS

S-CR II

0

TPP-El +

-E,

F 4 ) z . " 1 ,

0

II -E2

fi(CHJ,C

[ RCHO -TPP] -El

(7)

-I-

s-s 0 n ( C H & C . -II- % HS

S-CR II 0

SH

HS

+

o

HS-CoA

DPN

+

II

RC- S-COA

DPNH

+

Hi

R = CH, , HOOC (CH,), FIG.1. Reaction sequence in the CoA- and DPN-linked oxidative decarboxylation of a-keto acids.

thrcc csscIitid cnaymdtic component,s, designatccl lCl, E2,arid E, in Fig. 1 (Koike and Reed, 1901). It should bc cmphasizcd that reactioiis (7) through (11) occur within thcsc multicnzyme complexes. A high dcgrcc of spatial orientation of the enzgr-ncs within thc complcxcs must exist to permit interaction of the bound coenzymes (see Scctiori 111,A, 6).

BIOCHEMISTRY O F IJPOIC ACID

7

1. lkcurboxylution Reaction

The decarboxylation rcaction, Eq. (7), is visualized as a cleavage of the a-keto acid to yield COz and an enzyme-bound “aldehyde-thiamine pyrophosphate” (RCHO-TPP) compound, i.e., “active aldehyde.” There is now unequivocal evidence for this reaction since a pyruvic carboxylase (El) has been shown to be an essential component of the E. coli pyruvate dehydrogenation complcx (Koike and Reed, 1961; Gounaris and Hager, 1961) and the nature of the “aldehyde-TPP” compound has been elucidated (Breslow, 1958; Breslow and McKelis, 1959; Krampitz et al., 1961; Holzer and Beaucamp, 1961; Carlson and Brown, 1961). The occurrence of reaction (7) could be inferred from earlier observations. First, E. coli fraction A (a complex of enzymes El and Ez) catalyzes a TPP-dependent and CoA-independent exchange reaction between C1402and the carboxyl group of pyruvate (Korkes, 1953). Similar results have been obtained with the pyruvate dehydrogenation complex of pigeon breast muscle and the a-ketoglutarate dehydrogenation complex of pig heart (Goldberg and Sanadi, 1952). The pyruvate-C1402exchange reaction is insensitive to arscnite (Gounaris and Hager, 1961), thus indicating that lipoic acid is not involved in this reaction. Second, acyloin formation from pyruvate, particularly in the presence of addrd aldehydes, has been observed with the mammalian pyruvate dehydrogenation complex (Schweet et al., 1951) and with E. coli fraction A (Hager, 1953; Gunsalus, 1954a), and shown to be TPP dependent. Acyloiri synthcsis does not involve lipoic acid (O’Kane, 1950; Doliri and Gunsalus, 1951). Third, oxidation of pyruvatr and a-ketoglutarate with ferricyariide as oxidant, Eq. (la), observrd ItCOCOaH

+ 2Fe(CN)sa + HLO

3

RCOLH

+ COL + 2Fe(CN)6-4 + 2Hf

(12)

with the mammalian (Schwcrt ct al., 1951; Sanadi et al., 1952; Massey, 1960h) and bacterial complexes (Hager, 1953; Koike and Reed, 1960) requires TPP, but not CoA or DI”. Release of bound lipoic acid from the E. coli pyruvatc and a-krtoglutaratr dehydrogeiiation complexes did not affect their ability to catnlyzc reaction (12) (Koike and Reed, 1960). A further i ndi c a hn that bound lipoic acid does not participate in reaction (12) is the insensitivity of this reaction to arsenite (Sanadi et at., 1959b). These findings suggested that ferricyanide oxidizes the postulated RCHOTPP compound to RC02H. Fourth, it is to be noted that all known cleavages of a-keto acids require thiamine pyrophosphate, and an “aldehydeTPP” compound may be considered to be a common intermediate in the reactions, whether by straight decarboxylation, decarboxylation coupled with acyloin synthesis, or oxidative decarboxylation. Finally, Koike and Reed (1961) have resolved the E. coli pyruvate dehydrogenation complex

8

LESTER J. REED

and have demonstrated that the enzymatic component which catalyzes rcact2ion(12) is rcquircd in ovcr-all reaction (3). a. Nature uj’ “active alddqdc,.” Hapid progress in the elucidation of the mcchanism of thiamiric pyrophosphate action has followed from Breslom’s studies with a rionenzymatic model system (Breslow, 1958; Breslow and OH 0

R- CH2- N

CH,COCO,H

*

\

R-CH2-N

\

/c=c CH, \R’

/c=c\R‘

CH,

(IV)

(V)

-co,

qH

OH \ CH,CO

CH,C,H

\

R-CH2-

\

N

R- CH,-N,

\

/c=c

CH,

R=

.$-

A*

F=“\

CH,

‘R’

R’

=

’

R‘

CH,CH,OP,O~-

H3C FIG.2. Mechminm o f thiamine pyrophotspliitte action in the decarboxyl aLioii of pyruvate (Bredow, 1958).

McNelis, 1959).This subject hus been reviewed recently by Metzler (1960) and will riot be discussed in detail here. In brief, Breslow postulated that thiamine pyrophosphatc ionizcs a t the %position of the thiazole ring, and tliat the thiazolium dipolar ion (IV) reacts with pyriivate to form an intermediate (2-lactylthiamine pyrophosphate) (V) which undergoes decarboxylation to produce 2-hydroxyethylthiamine pyrophosphate (VI, VII) (Fig. 2). Species (VI) is regarded as “active acctaldehyde,’’ and can

9

HIOCHEMISTHY O F LIPOIC ACID

give rise to acetaldehyde, acetoin and acetolactatc as illustrated in Eqs. (13-15), respectively.

-

(VI)

2 YH

CH,C-CHCH,

I

0

I1

CH,CH

(13)

+ (rV)

(Iv)

HO OH I I CH,Y-CHCH,

(VI)

+

0 II CH,CH-

R-CHZ-N

(14)

0F-7 \

/c=c CH, 'R'

HP YH CH,C-C(CHJ 6-S (VI)

t

CH,COC02H ___ - R - c H r % c

I F""\

CH,

'I

CH,C-C0 II OH CO, @ CH,

+

(Iv)

CO? (15)

R'

_1

Several groups of investigators have independently obtained evidence which supports and amplifies this mechanism for the enzymatic processes. Krampitz et al. (1961) synthesized 2-hydroxyethylthiamine pyrophosphate (VII) and demonstrated that the hydroxyethyl group undergoes (a) conversion to acetaldehyde in the presence of wheat germ carboxylase and (b) conversion to acctoin in the preserice of the acetoin-forming system from Aerobacter aerogenes. 2-Hydroxyethylthiamine pyrophosphate was

isolated from reaction mixtures of wheal germ apocarboxylase, ‘L’PP, and pyruvute, as well as from mixlurcs of the A . ucrogenes acctoin-forming system, TPP, arid pyruvutc. Iiolzer and co-workers (Holzer and Beaucamp, 1961; Holzer et aZ., 1960; Scriba and Holzer, 1961; Holzer and Kohlhaw, l!Kjl) isolated 2-laetylthiamine pyrophosphate and 2-hydroxycthylthiairiiiic pyrophospliatc from reaction mixtures of yeast pyruvic carboxyltihe arid pyriivatc and also isola tcd 2-hydroxyelhylthiamine pyrophosphate from rcuction niixturcv of pyruvate :ind the pyruvate oxidation system of yeast mitochondria and of pig heart, musclc. Conversion of the hydroxyrthyl group of 2-hydroxyethylthinlviirie pyrophosphatc to acetaldchydc, acctoin, and ucctolactate was demonstrated in these enzyme systems. Carlsou and Brown (1961) demonstrated the natiiral occurrence of 2-hydroxyethylthiaminc pyrophosphatc in microorganisms. They also isolated this compound from reaction mixtures of wheat germ apocurboxylase, TIT, and pyruvate or uccbldehydc, and demonstrated that the hydroxycthyl group is convcrtcd to acetoin in this system. 2. Acyl-Conmxtion Reuclion

Thc ncyl-grneration reaction, Ey. (8), has been visualized a5 a reductive arylation of protein-bound lipoic acid. As will be aren below, this rcJaction is now belirvcd to consist of two steps: an oxidatioii of the 2-hydroxyalkylt hiarniric. pyrophosphatc. to 2-arylt hiuininc pyrophosphatc with n concomitant rrduction of bound lipoic. acid, and L: trtinsfcr of the twyl group of 2-ac~ylthiaminepyrophosphate to t he bound dihydrolipoic acid (Ilas ct ul., lS(i1). An enzymatic component which contains bound lipoic acid and apparently catalyzes reactions (8) arid (1)) has been isoltiCcd from the E . coli pyruvatc dehydrogenation complex (Koike arid Reed, 1961). This component, designated lipoyl-Ez in Fig. 1, has been tentatively named lipoic reductase-transacetylase. Gunsalus tirid co-workers (Gunaalus, 1954b; Gunsiilus and Smith, 1958) adduced evidence for reaction (8) from reactions obtained with substrate amounts of lipoic acid. They reported that a small amount of a thiocstcr, prehurricd to be S-acctyldihydrolipoic acid, was produced when E. coli fraction A was incubated with pyruvatc, TPY, and a substrate amount of lipoic acid (Lipss). When the incubation mixture was supplemented with phosphotransacetylase, inorganic phosphate, and a catalytic amount of CoA, substantial and equivalent amounts of acetyl phosphate, dihydrolipoic acid (Lip(SH)s] and COz were produced [Eq. (lri)]. Them results CHICOC02H

+ Lips2 + Hl’Oa-

(COA)

CHI COOI’O3- -

+ Lip (YH), + COL

(16)

were interpreted as indicating a reductive acetylation of external lipoic acid, followed by acctyl transfer to C o h and then to phosphate. A detailed

BIOCHEMISTRY OF LIPOIC ACID

11

analysis of reaction (16) by Reed and co-workers (Reed et al., 1958b; Koike and Reed, 1960) revealed requirements for protein-bound lipoic acid and flavoprotein (E, in Fig. 1).The reaction sequence therefore appeared to be more complicated than that visualized by Gunsalus and co-workers. Reed and coworkers interpreted their data as indicating that the sequence in reaction (16) is reactions (7) through (10) involving protein-bound lipoic acid, followed by reduction of the external lipoic acid by the reduced flavoprotein. I n other words, external lipoic acid functions as the ultimate electron acceptor. The acetyl CoA generated in reaction (9) can lead to acetyl phosphate through the phosphotransacetylase-catalyzedreaction [Eq. (17)]. CHJX-S-CoA

+ HPO4- - + CHaCOOPOx- - + COA-SH

(17)

The production of a small amount of AS-acetyldihydrolipoic acid can be attributed to interaction of acrtyl CoA and dihydrolipoic acid, catalyzed by dihydrolipoic transacet ylase. A similar interpretation of the data of Gunsalus and Smith was presented by Sanadi et al. (1959b). Koike and Reed (1960) were unable to demonstrate production of Sacetyldihydrolipoic acid (or S-acetyldihydrolipoamide) from pyruvate and external lipoic acid (or lipoamide) with the highly purified E. coli pyruvate dehydrogenation complex. Sanadi et al. (1959b) also reported failure to demonstrate this process with E. coli fraction A; nor could they demonstrate production of S-succinyldihydrolipoic acid from a-ketoglutarate and external lipoic acid in the presence of the pig heart a-ketoglutarate dehydrogenation complex. However, Goldman (1960) has reported the production of S-acetyldihydrolipoic acid from pyruvate and external lipoic acid in the presence of a partially purified preparation of the Mycobacterium tuberculosis pyruvate dehydrogenation complex. Production of S-acetyldihydrolipoic acid was inhibited by N-ethylmaleimidc, but not by arsenite, and CoA did not appear to bc involved. The reaction was specific for (+)-lipoic acid. Goldman assumed that protcin-bound lipoic acid participates in the rcaction and proposed that the extcrrial lipoic acid interacts with the proteinbound S-acetyldihydrolipoic acid [generatcd in reactions (7) and (S)] a s illustratcd in Fig. 3. This scyucnce involves thiol-disulfide interchange reactions a s wcll as an acctyl transfer reaction. A similar reaction sequence had been suggested previously by Reed et al. (1958b) as a possible explanation of the results of Gunsalus and co-workers. The conflicting results obtained with substrate amounts of lipoic acid probably reflect differences in the abilities of the various enzyme preparations to couple with external lipoic acid. The mechanism of these coupled reactions is in need of definition. Chin and Gunsalus (1954) adduced evidence for reversal of reaction (8) from the observation that a w r y small amount of acetoin was produced in a rcsction mixture containing E. coli frartion A, phosphotrarisacetyl,zsr,

12

LESTER J. REED

CoA, TPP, acctyl phosphate, acetaldehyde, and a substrate amount of dihydrolipoic acid. The results were interpreted as indicating conversion of acetyl to “scctaldehyde-TPP” by reversal of reaction (8), followed tiy reaction of ncetaldchyde with the “acetsldchyde-TPP” compound to prod i m acetoin. Use of u ~ e t , y l - b Cphosphate ~~ confirmed the incorporation of acetyl irito aretoin. The results of Chin and Gunsalus should be considered indicative rather than proof, and further analysis of the reaction sequence is desirable. The complexity of reaction (16) suggests caution in interpreting thcir results.

tl

R = (CH,),CO,H

R’ = (CH,),CO-enzyme

FIG.3. Reductive acetylation of external lipoic acid by S-acetyldihydrolipoy enzyme ((hldmnn, 1060).

It should be appsrcnt, from t,hc discussion thus far tdiat external lipoic x i d cannot, rcplaoe l,hc c:at~alytticallyactive protoin-hound lipoic acid in nay1 gcwr.:it>ionfrom a-kclo acids. The results ohtJ:iined with sutwtjrat,e :miowits of rxtcrnal lipoic acid therefore do not provide unequivocal evidence for r e s ot hi (8). The cvidencc presentd by Sanadi ct al. (195%) is ixwre sat,isf:ictory in this respect. These investjigstors isolated CY-labeled succiriohydroxaniic acid from an incubation mixture containing a large excess of pig heart a-ketoglutarate dehydrogenation complex, a-ketoglutarate5-C14, and hydroxylamine. The enzyme preparation did not contain CoA. These results were interpreted as indicating t,hc formation of a reactJim succinyl group which is bound tjo the enzyme complex. Sanadi et al. (1059b) &o reported that preincubstion of the cw-ket,oglutarat,edehydrogenation complcx wit,h nrsenite in the presence of both a-ketoglut,nrate and CoA re-

BIOCHEMISTRY O F LIPOIC ACID

13

sulted in inhibition of over-all reaction ( 3 ) . If it is assumed that the site of arsenite inhibition is the protein-bound dihydrolipoic acid, the requirement for both a-ketoglutarat,e and CoA to produce the inhibited complex is consistent with reactions (8) and (9). The CoA requirement also would appear to preclude attachment of the reactive succinyl to a site on the enzyme complex other than the bound dihydrolipoic acid. a. 2-Acylthiamine pyrophosphate as intermediate. Further insight into the nature of reaction (8) was gained by resolution of the E. coli pyruvate dehydrogenation complex and by a logical extension of Breslow’s mechanism of thiamine action (Das et al., 1961). The pyruvic carboxylase component of the E. coli pyruvate dehydrogenation complex catalyzes a fcrricyanidelinked oxidation of pyruvate [Eq. (la)]. Since the only cofactor involved in this reaction is TPP, it appeared that ferricyanide reacts with the postulated “active acetaldehyde” as represented by Eq. (18). Reactions (8) and (18), however, presented an inconsistency. When enzyme-bound lipoic acid serves as oxidant, for “active acetaldehyde” an energy-rich acetyl com[CHICHO-TPP]-EI

+ 2Fe(CN)6--- + HzO CH:
(18)

pound, enzyme-borind S-acetyldihydrolipoic arid, is produced [Eq. (S)], whereas when ferricyanidc serves as oxidant, a n energy-poor acetyl compound, acetate, is produced (Eq. (IS)]. Since the same enzyme participat,es in reactions (8) and (18), it seemed likely that a n energy-rich acetyl compound should be an intermediate in both reactions. That this energy-rich acetyl compound might be 2-ac~hylthiarninepyrophosphate follows logically from Breslow’s proposal concerning thc mechanism of thiamine action. Recent publications by Breslow and his co-workers pointed u p this possibility. Breslow (1959) postulated that 2-acetylthiamine pyrophosphate is an intermediate in tlhe phosphoketolase-catalyzed reaction of xylulose-5phosphate with inorganic phosphate t,o give glyceraldehyde-3-phosphate and acetyl phosphate (Heath et al., 1958). Implicit in this suggestion is the requirement that 2-acetylthiamine pyrophosphate be able to acetylate phosphate ion and thus be yet another form of ‘lactive acetate.” I n support of this prediction Breslow and McNelis (1960) reported the preparation in crude form of 2-acetyl-3,4-dimethylthiazolium nitrate and presented evidence that this compound is easily deacylat,cd in the presence of water or methanol. White and Ingraham (1960) reported similar observations indicating kinetic instability of a crude prcparat,ion of 2-benzoyl-3,4-dimethylthiazolium iodide. Das et al. (1961) were able t,o demonstrat,e the formation of acetyl phosphate in the ferricyanide-linked oxidat’ion of pyruvate catalyzed by the E. coli pyruvic carboxylase. In this system phosphate ion serves as a trap for

14

LESTER J. REED

t,he “a(4ive” acetyl groups. These results can bc represented by an extcnsion of Brcslow’s proposed mechanism (cf. Fig. 2) as illustrated in Fig. 4. Oxidation of enzyme-bound 2-hydroxyethylthianiine pyrophosphate (VI) by fcrricyuriide produces 2-acetylthiamine pyrophosphatc (VIII). Nucleophilic attack of water or dihydrogen phosphate ion on the carbonyl carbon atom of 2-acetylthiamine pyrophosphate produces acetic acid and acetyl phosphate, rcspect,ively. Presumably these are competing reactions since the proportion of pyruvatc converted to acctyl phosphate increases with increasing concentration of phosphate buffer. Das et al. (1961) also presented evidence that succinyl phosphate is produced in the ferricyanideliriked oxidation of a-ketoglutaratc catalyzcd by the B. coli a-ketoglutarate OH \ CH,C,@

-

2 F e (CN),--

R- CH2- N / CH,

0 \\ CH,C

+

H’

2 Fe (CN):-

t

/c=c‘R’

\

CH,

R’

(VI1

( VIII )

0 ii

HOH

CH,C-OH 0

II CH,C-OPO,

I

__

H’ I

I

(TV)

2 Hi

1

(IV)

FIG.4. Mechanism of thiainine pyropliospliat,e action in thc fcrricynnide-linked oxidative dccarboxylntion of pyruvntc ( D n s et al., 1961). The decarbosylation process is illustrated in Fig. 2.

dehydrogenation complex, when phosphate is present in the reaction mixture. Presumably, succinyl phosphate is formed by nucleophilic attack of dihydrogen phosphate ion on 2-succinylthiamine pyrophosphate (IX). H,C-

R- CH,-N

/

+-I

O=F

CH, \

c\=8

O F=C \ CHs R’ \

(1x1 Daigo and Reed (1962a) cxtcndcd the work of Breslow and McNelis (1960) and White and Ingraham (1960) and obtained evidcncc for thc

BIOCHEMISTRY O F LIPOIC ACID

15

thermodynamic instability of 2-aretylthiazolium salts. They synthesizcd the model compound 2-acetyl-3,4-dimcthylthiazolium iodide (X) and pre”1

I

SR

0

CH,C=O

$/C=CH 7

H,C -N

\

H,C-N 0F-Y \

/C=CH

+

0

II CH,C-SR

(19)

I@ CH,

I@ CH,

(X) sentcd evidence that this compound undergoes nucleophilic attack by water to give acetic acid, by hydroxylamine to give acethydroxamic acid, and by mercaptide ions to give thiolacetates. The lat,ter reaction, Eq. (19), was obtained with the mercaptide ion of dihydrolipoamide. These observat,ions suggcst that 2-acetylthiamine pyrophosphate is a n intcrmediate in the oxidative decarhoxylation of pyruvate involving the [CH,CHO-TPP]-E,

-1

0 n ( C H , ) , C - - IIE ,

s-s

16

LESTER J. HEED

physiological oxidant,, cnzyme-hound lipoic acid. The oxidative reaction, Eq. ( 8 ) , would appear to proceed in two strps [reactions (20) and (2l)l. Presumably these arc tightly coupled reactions, in view of the kirictic lability of 2-acetylthia~oliiir~i salts arid the observatiori of Sariadi P t al. (195%) that CoA as well as a-ketoglutaratc is required for arsenite inhibition of the a-krtoglutamte dehydrogenation complex. The rate of reaction (21) rnust be very rapid to preclude hydrolysis of 2-acetyli hi:imine pyrophosphate and also inhibition of thc dihydrolipoyl enzyme by arsenite. That 2-acetylthiamine pyrophosphate is an intermediate in the oxidative decmboxylutiori of pyruvate may also be inferred from (a) the demonstration by Goedde et a/. ( I 961) that the hydroxyethyl group of 2-liydroxyethylthiamine pyrophosphatc can he converted to acetyl CoA in the prcscriche of a pyruvate oxidation system from yeast, mitochondria and (b) the demonstration by Krarnpitz P t a/. ( I 961) that the hydroxyethyl group of 2-hydroxyethglthiamint pyrophosphutc is oxidized to ncctatc iii the presrilrr of fcrricyariidc arid the acetoin-formitig system from A . arrogmcs. I t appears reasonable to suggest that 2-acetylthiamirie pyrophosphatc is ari iritermcdiate in all pyruvkztc oxidation systems, and possibly in the various “c1:istic” clcavugcs of pyruvatr as well (see Gunsalus et al., 1955, for a rcview of these latter rrac.tions). A generalized mechanism uf pyruvatc oxidation vibualixcs (a) dccarboxylation of pyruvtitc to give 2-hydroxyethylthiamine pyrophosphatc, (b) oxidation of the latter compound to 2-acetylthiamiric pyrophosphatc, and (c) acctyl tratisfcr to a thiol, phosphate, or water. The oxidizing agent iriay bc protciii-boiind lipoirh acid, t ~ 1s11 thc ( h A and DPN-linked a-keto acid dehydrogcn:ttion complcxcs; FA I), as iri thc classic l,acto/)acillus dclbrueckii pyruvic oxidase ( Lipmarin, 1999; Hager et al., 1!)54) arid in the cytochromr-linked pyruvic oxidase from I’TO~CUSvulgaris (Moycd arid O’I
17

BIOCHEMISTRY O F LIPOIC ACID

thiamine pyrophosphate appears to be tightly coupled with acetyl transfer to a thiol or to phosphate. In the CoA- and DPN-linked pyruvate dehydrogenation complexes, the oxidizing agent is also the acetyl acceptor, i.e., lipoyl-Es, and a tight coupling of oxidation and acetyl transfer is easily visualized. A tight coupling of oxidation and acetyl transfer also appears to exist in the L. delbrueckii pynivic oxidase. The latter is a TPP-FAD enzyme which oxidizes pyruvatc to acctyl phosphate and CO2 (Lipmann, 1939; Hager et al., 1954). Xcit>herlipoic acid nor CoA is involved in the rcaction. Hager and Lipmann (1961) recently reported that the enzyme shows an absolute phosphate requirement for 14’AD reduction by pyruvate. They visualize a concerted reaction, wilh FAD pulling electrons off the hydroxyethyl group of 2-hydroxyethylthiamine pyrophosphate while nucleophilic attack by phosphate iori on the a-carbon atom displaces thiamine pyrophosphate. The niechanisni of the "elastic" cleavages of pyruvate, producing acetyl phosphate and formate or COZ and Hz,is still obscure. Biotin (Schuster and Lynen, 19GO), folic acid (Delavier-Klutchko, 1959), and vitamin B12 derivatives (Rabinowitz, 1960) have recently been implicated in these rcactions. Itl scems possible that these may also be examples of tightly coupled systems in which 2-acetylthiaminc pyrophosphat,e is an intermediate. 3. Acyl-Transfer Reaction

The acyl-transfer reaction, Eq. (9), is visualized as an acyl exchange between protein-bound S-acyldihydrolipoic acid and CoA to give proteinbound dihydrolipoic acid and acyl CoA. Evidence for this reaction is based on model reactions carried out with substrate amounts of dihydrolipoic acid and on the arsenite inhibition studies of Sanadi et al. (195913) discussed in Section 111, A, 2. An enzymatic component, lipoic reductasetransacetylasc, which apparently catalyzes reactions (8) and (9), has been isolated from thc A’.coli pyruvate drhydrogenation complcx (Koikc and Reed, 1961). It is uncertain whether this cornponcnt consists of one or two enzymes. Hager and Giinsalus (Ilager, 1953; Gunsalus, 1954a) observed that incubation of fi. coli fraction A and phosphotransacctylase with acetyl phosphate, m-dihydrolipoic acid, and a catalytic amount of CoA resulted in production of a thioester and disappearance of an equivalent amount of thiol. This observation was interpreted as indicating a coupling of the phosphotransacetylase reaction, Eq. (17) (reverse direction), and reaction (22) to give reaction (23). Reaction (22) was attributed to a n enzyme, dihydrolipoic transacetylase, present in B. coli fraction A. Since not more than CH3CO-S-CoA

+ Lip(HH)z

4

CHaCO-S-Lip--SH

+ CoA-SH

(22)

18

LESTER J. REED

CH3COOP03- + Lip (SH)2

(CoA)

’ HPOI- - + CHaCO-S-Lip-SH

(23)

one-half of the m-dihydrolipoic acid was utilized, even in thc presence of excess fraction A, it was concluded that dihydrolipoic transacetylase exhibits optical specificity. These results have been confirmed by Reed et al. (195813) and by (:oldman ( I 959). Further evidence of specificity is furnished by the finding that dihydrolipoic acid cannot be replaced in reaction (23) hy glutathione, mercaptoethanol, thioglycolic acid, or N ,N-diethylmercaptoethylamine (Sanadi et al., 195%). Gunsalus et al. (1956) rcported that ( - )-dihydrolipoic acid (prcpared by chemical reduction of (+)-lipoic acid), hut not (+)-dihydrolipoic acid, is acetylated in reaction (23). They isolated the thioester and characterized it H2

~CF~CH(CI~,),CO,H

I

HS

I

SCOCH,

6-S- Acetyldihydrolipoic acid

(XI1

as (+)-6-S-acetyldihydrolipoic acid (XI). Gunsalus and Smith (1958) reported that a niono-S-succinyldihydrolipoic acid was produced enzymatically by coupling succinic thiokinase and E. coli fraction A’ (derived from the a-ketoglutarate dehydrogenation complex). Succinic thiokinase generates succinyl CoA [Eq. (24)], and transfer of the succinyl group to dihydrolipoic acid [Eq. (25)] is catalyzed by dihydrolipoic transsuccinylase (present in fraction A’). Although details of the biosynthesis and characH02C(CI-I2)&02H

+ ATP + COA-SH

+

~

-

f

HOAC(CII~)?CO-S-COA

+ ADP + HPOd--

HOtC(CHJjC0-S--CoA Lip(8H)z -+ H02C(CH2)LCO-S-Lip-SH

+ CoA-SH

(24)

(25)

terization of the mono-l’i-succinyldihydrolipoic acid were not reported, thc d a t t ~presentrd indicaatc that cnzymatic transsucciriylatiori of dihydrolipoic.

avid also occurs on the secondary sulfhydryl group, is ., a t C-6. A coupling of reactions (24) and (25) to produce a hydroxylamine-reactive substance, presumably Ssuccinyldihydrolipoic acid, has been noted by Hager and Kornberg (1961) with extracts of 23. coli (strain W) and by Reed and Koike (unpublished results) with a purified preparation of succinic thiokinase and the highly purified E. coli a-ketoglutarate dehydrogenation complex. IIo\vever, Sanadi et al. (1959b) have reported failure in attempts to produce S-suc.c’inyldihydrolipoic acid by cbonplirig suctinic. t hiokinase and t he pig

UIOCHEMISTHY OY LIPOlC ACID

1‘3

heart a-ketoglutarate dehydrogcnat,ion complcx. A satisfactory explanation of this discrepancy is not apparent a t present. Koike et al. (196Oa) reported that the ratio of dihydrolipoic transacetylase activity, as measured by reaction ( 2 3 ) , to pyruvate dismutation activity [based on reaction (3)] was constant over a 250-fold range of purification of the E. coli pyruvate dehydrogenation complex. Recently, Koike and Reed (1961) reported thc isolation from t,hc latter complex of the component which catalyzes reaction ( 2 3 ) . This component, lipoic reductasc-transacetylasc, contained all the protein-bound lipoic acid present, in the complex and was required to reconstitute the over-all pyruvate dchydrogenation activity [reaction (3)]. Koike and Reed (1960) had observed previously that release of approximately 96 3’2 of thc bound lipoic acid from the I?. coli pyruvatc dehydrogenation complex did not affect the dihydrolipoic transacetylase activity, as measured by reaction (23), whereas the over-all pyruvate dehydrogenation activity [reaction (3)] was markedly decreased. Reincorporation of lipoic acid into the complex by means of the lipoic acidactivating system restored the latter activity, but the dihydrolipoic t,ransacetylase activity was not affected. These results demonstrate that thc protein-bound lipoic acid does not part,icipnte in the model transacetylation reaction, Eq. ( 2 3 ) , carried out wit,h a subst,rate amount of free dihydrolipoic acid. It may be inferred from these results that lipoic reductastransacetylase is a complex of t,wo separate enzymes-a rediictase which contains t,he bound lipoic acid and catalyzes reaction (S), and a transacetylase which catalyzes the physiological reaction, Eq. (9), involving the bound cofactor, as well as the model reaction, Eq. (22), involving free dihydrolipoic acid. However, an alternative possibility is that lipoic reductasctransacetylase is a single enzyme possessing two active centers, i.e., a “double-headed” enzyme. Further study of this component should distinguish between these two possibilities. It should be emphasized that the transacylation reactions represented by Eqs. (22) and (25) have been carried out under nonphysiological conditions, i.e., with substrate amounts of dihydrolipoic acid. The physiological reaction, Eq. (9), involves a catalytic amount of covalently bound lipoic acid. It does not necessarily follow, therefore, that the acetyl and succinyl groups generated from pyruvate and a-ketoglutarate, respectively, are linked to the secondary sulfhydryl group in the bound dihydrolipoic acid. Moreover, the difficulty of obtaining the S-acyldihydrolipoic acids enzymatically in pure form and in adequate quantity has limited evaluation of their ability to serve as acyl donors (Goldman, 1959, 1960). Gunsalus and Smith (1958) reported that arsenolysis of S-acetyldihydrolipoic acid [produced enzymatically in reaction (23)l occurred in the presence of E. coli fraction A,

20

LESTER J. l t E E U

phosphotr:tns:Lc~i~ylasc, imcriatr, and a catalytic amount of CoR. This observation was interpreted as indicating ~ Kacctyl I trmsfer from S-acet,yldihydrolipoic acid to CoA, i.e., a reversal of reaction (22). It was noted, howcvcr, that the rate of arsenolysis was not strictly proportional to fraction A roncentration and was slower than the ratr of acetyl transfer in the oppositcb dircct ion [reaction (%<)I.

4. Electron- Transfw Rcactioii ( )xidation of protein-bound dihydrolipoic acid [15q. (1 O)] is :wcomplkhed hy a11 I~rZI)-fl:1Loproteiii, dihydrolipoic dchydrogenilsc. This cnzyme has I m n separated from the other cnzynirs comprising the a-keto acid dehydrogenation cornplcxcs and its mechanism of artion has been the subject of intensive investigation. As will be seen below, thcrc is good evidence that the pig heart dihydrolipoic dehydrogenase rontains a reactive disulfide group, presumably that of a cystine residue, which participates in the cat,alytic cycle. A biradicnl form of the reduced Aavoprotcin, comprising a sulfur radical and a flavin scmiquinonc, is apparrnlly an intermediate in the over-all t wo-elect roil t ransfer from protein-bound dihydrolipoic acid to DPiS [b:qs. (10-Il)](RiIassc~yand Veeger, 1961; Scarls ct al., 1961). The classic studies of Pctcrs and co-workers (Peters ~t al., 10.16; Stocken and Thonipsoil, I94C,a, b) showing inhibition of the br:tin pyruvate oxidation system hy trivulerit urscnicnls a n d reversal by the dithiol, 2,3-dinicrcaptopropanol (HA l,), hiit not, by monothiols Icd t hehc investjigstors to postulate the involvcmcnt of a dithiol structure in pyruvatc oxidation. I t is now known that t hix dithiol structure is covalently bound dihydrolipoic acid. The latter substance apparently forms n stnble vyrlic thioarscnite with trivalcnt :wxciiirals, a n d therrby its (~atalytitaction in a-keto acid oxidation is inhibitrd. I t is prrtincnt to iiotc that Iteiss (lU58) has shown that, y-(p-arsenosophcriyl)-n-butyrate, a potcnt inhibitor of pyriivate oxidation by rat heart sarrosomcs, cornbincs with dihydrolipoir acid in vitro in a I :1 molar ratio. Separation of the It. coli pyruvate and a-ketoglutarate dehydrogenation systems into thrcc protein fractions one (A) spccific for pyruvate and a second (A’) specific for a-ketoglutarate, plus a third fraction (B) common to the two systems-suggested a dehydrogcnase function for fraction B (Hager and Gunsalus, 1953). Strong support for this conchision was provided by the demonstration thal fraction H catalyzed the oxidation of dihydrolipoic acid by DPN [b:q. (%)I. Dihydrolipoic dchydrogenase activity was shown

I,ip(SH)L

+ UPN

Lips,

+ I)PNlI + H+

(26)

subsequently to br associated with the pig h c w l a-ketoglutarate dchydrogenation complrx (Sanadi arid Searls, 19,1,7; Sanadi et al., 1959a), and dihy-

BIOCHEMISTRY O F LIPOIC ACID

21

drolipoic dehydrogenases were purified t,o varying degrees from extracts of Leuconostoc mesenteroides (Notani arid Gunsalus, I9;i8), AT. tuberculosis (Goldman, lSriU), and spinach ( B ~ Wand Burma, 1960). Sanadi et al. (1‘35%~)reported that, reaction (26) is freely reversible wit,h either DLlipoic acid or m-lipoamide in the presence of tho pig heart a-ketoglutarate dehydrogenation complex. The equilibrium constant of the reaction was determined, permit,t,ingcalculation of the reduction potential of the Lip(SH)zLipsz system (see Section 11, A). The apparent K , values for lipoic acid and its derivatives in reaction (26) are approximately to M (Sanadi et al., 1959a; Massey, 1960a). These values are larger by several orders of magnitude than the amounts of bound lipoic acid required for comparable rates in the physiological react.ion, Eq. ( 3 ) (Sanadi et al., 1959a; Koike et al., 1960s; Massey, 19BOb). Reed and co-workers (Reed et al., 1958b; Koike and Reed, 1960) showed that release of bound lipoic acid from the 8.coli pyruvate and a-ketoglutarate dehydrogenation complexes did not, affect the abilit8yof these preparations to catalyze react.ion (26). Analyses of highly purified preparations of dihydrolipoic dehydrogeriasc from bacterial (Hager, 1953; Koike et al., 1960b), animal (Massey, 196Ob; Searls and Sanadi, 196Oa), and plant (Matthews and Reed, unpublished result’s) sources have demonstratcd thc absence of bound lipoic acid. It is clear therefore t.hat the bound lipoic acid in t,he complexes does not participatc in the model react,ion, Eq, (26). I n other words, dihydrolipoic dehydrogenase can react with the bound cofactor (dihydrolipoyl-Ez in Fig. 1) as well as with free dihydrolipoic acid and its derivatives. A significant advance in understanding of the mechanism of the dihydrolipoic dehydrogenasc reaction resulted from the discovery by Massey (1958) that the classic flavoprot’ein first isolated by Straub (1939), and widely known as “St’raiib’s diaphorase,” behaves as a powerful dihydrolipoic dehydrogenase. Diaphorase activity was measured with ferricyanide as elect,ron acccptor, Ey. (27), and dihydrolipoic dehydrogenasc activity by DPNH

+ 2Fe(CN)s---

4

1)PN f 2Fe(CN)64- f Ht

(27)

reaction (26), from right, t’o left. Evidence was adduced that these two activities are associated with the same enzyme. Sanadi et al. (1052) had reported previously that highly purified preparations of the pig heart a-ketoglut’arate dehydrogenation complex exhibited diaphorase activity, but attributed this activity to an impurity in their preparations. Massey’s discovery suggcsted that diaphorase is an integral part of the complex, i.e. that dihydrolipoic dehydrogenase is a flavoprotein and is identical with Straub’s diaphorase. This conclusion was confirmed (Massey, 1959, 1960a, b) by resolution of the complex into a colorless fract’ion (comprising

22

LESTER J . REED

enzymes El and &, Fig. 1) arid a flavoprotein which was shown to be idcntical in physicochernical and enzymatic properties with Straub’s diaphorase. The report of NIassey (1!)58) that Straub’s diaphorase is a powerful dihydrolipoic dehydrogenase was soon followed by reports from other laboratories indicating that dihydrolipoic dehydrogenase is a flavoprotein. Rccd and co-workers ( b i k e and Reed, 1959, 1960; Koike et al., 1960a) reported that highly purified preparations of the h’. coli pyruvate and a-ketoglutarate dehydrogenation complexes contained significant amounts of FAD. Release of PAD by treatment of the complexes with acidic ammonium sulfate resulted in a marked decrease in pyruvatc and a-ketoglutarate dehydrogenation activity [Eq. (3)] and in dihydrolipoic dehydrogenase activity [Eq. (2A)I. Thcse activities were largely restored by addition of FAD. Purification of 13. coli fraction H (Hager and Gunsalus, 1953) was extended t o obtain an essentially homogeneous preparation of dihydrolipoic dehydrogenase, and the latter enzyme was shown to be an FAD-flavoprotein and to exhibit powerful diaphorase activity [Eq. (27)] (Koike et al., 1960b). Searls and Sanadi ( I 959, 196Oa) diaruptcd thc pig heart a-ketoglutarate dehydrogenation complex by digrstion with trypsin, isolated the componcnt which catalyzed reaction (26), and showed it to bc an FAD-flavoprotein. The physical, enzymatic, and catalytic properties of this cnzyrnc and Straub’s diaphorasc were corriparcd and found to be very similar (Searls and Sanadi, 1961). As obscrvrd hpcctrophotomctricilly, the flavin of the pig heart a-ketoglutarate dehydrogenation romplcx is rcduccd by a-ketoglutarate CoASH and is rcoxidizcd by DPN (Massey, 196Ob). This reduction is inhibited by arsenite. The flavin in the E. roli pyruvatc and a-ketoglutarste dchydrogeriation complexes also was shown (Koike and Reed, 1960) to bc reduced hy pyruvate CoASH arid by a-kctoglutarate CoASH, rebpcctivcly. Ncithckr a-keto acid nor CoASI 1 alone caused reduction of thc flnvin. These rrhulta are corisistenl with thc scqucnce of reactions (7) through (11). Searls and Sanadi (1960s) determined the reduction potcntial of the pig heart dihydrolipoic dehydrogenase from the extent of its reduction ut different DPNH :DPN ratios:. Thc valuc is bctwecn -0.332 and -0.320 volt a t pH 7.0 and 23°C;. Thus the reduction potential of the flavoprotein is close to that of thc DPNH-DPN system, -0.320 volt a t pH 7.0 and 25°C (Burton and Wilson, 1953), and the Ilip(SH)z-llipSz system, -0.325 volt ut pH 7.0 and 25°C (see Section 11, A). This is consistent with the ready revcrsibility of reaction (26) as well as the high initial reaction rates in both directions. I t would appear that DPN is the physiological electron acceptor for the a-keto acid dehydrogenation complexes in viva and that the DPNH formed is reoxidizcd by way of the electron transport chain. However, the

+

+

+

BIOCHEMISTRY OF LIPOIC ACID

23

possibility must also be considered that the complexes are linked directly to the electron transport chain, possibly to a flavoprotein or to cytochrome b, without mediation of DPN. This possibility has yet to be investigated. Searls and Sanadi (1959, l960a) and Massey et al. (1960) observed an increase in absorbancy in the region between 500 and 600 mp (maximum a t 530 mp), concomitant with a decrease a t 455 mp, on reduction of pig heart dihydrolipoic dehydrogenase with DPNH and with dihydrolipoic acid.A similar effect was noted previously by Savage (1957) on reduction of Straub’s diaphorase with DPNH. These results recalled similar observations by Beinert (1957) with other flavoproteins which were attributed to the formation of a flavin semiquinone. Massey et al. (1960) have made a detailed study of the stoichiometry of formation of the 530-mp band and the kinetics of its formation and disappearance under a variety of conditions, and attributed it to a flavin semiquinone which is a n obligatory intermediate in the catalytic cycle of the enzyme. Addition of p-chloromercuriphenyl sulfonate to the partially reduced flavoprotein resulted in disappearance of the red color and further reduction of the flavin. This observation was interpreted as indicating that the flavin semiquinone is stabilized by interaction with a protein sulfhydryl group. A further advance in understanding of the mechanism of the dihydrolipoic dehydrogenase reaction resulted from the independent observation of Searls and Sanadi (1960b) and Massey and Veeger (1960) that pig heart dihydrolipoic dehydrogenase is inhibited by arsenite in the presence of reducing substrate, i.e., DPNH. The inhibition was reversed by BAL and to a lesser extent by monothiols (Searls and Sanadi, 1960b; Searls et al., 1961). The 530-mp band associated with the partially reduced enzyme was eliminated on addition of arsenite and further reduction of the flavin was observed. Analysis of the enzyme for -SH content with p-chloromercuribenzoate showed the appearance of 2 moles of -SH per mole of enzyme on reduction with D PNH (Searls et al., 1961). On the basis of these observations and other extensive spectrophotometric data (Searls and Sanadi, 1960a; Searls et al., 1961; Massey and Veeger, 196l), both Sanadi and coworkers and Massey and co-workers have concluded that pig heart dihydrolipoic dehydrogenase contains a second prosthetic group, i.e., a reactive disulfide, which participates in the catalytic cycle IEys. (10-ll)]. These investigators visualize a reduction of the prosthetic disulfide group by dihydrolipoyl enzyme to give FAD--Ea-(SH),, followed by interaction of the dithiol and flavin to give the intermediate responsible for the 5c
2-1

LESTER J. REEL)

show a signal in the RSK spectrometer because of electron sharing between the sulfur. mdical and thc flaviii semiqixinone. Scarls el al. ( 1 961) have ~tlso corisidcred the possibility that the intermediate may be a charge transfer complex. Recent studies by Msssey (1 96Oc) iiidicatc that the prosthetic disulfide group also plays an important role in maintaining the intact tertiary structure of the enzyme. It appears l o be a cystiiie residue holding together two polypeptide chairis (Massey and Veeger, 1961). Recent studies with the E . coli dihydrolipoic dehydrogenase have shed some light on its anomalous behavior. As noted previously (Hagcr, 1953; Koike ct al., 19GOb), the model rmction, Eq. (a(;),is not freely reversible with this enzyme, in contrast to pig heart, dihydrolipoic dehydrogcnasc. The E. coli enzyme is apparently inhibited by excess DPNH (Notani and (iunsalus, 1959; Koike ~t al., 1960b). Matthews and Reed (unpublished results) have obswvcd that this inhibition is prevented by DPN. Massey and Vecger (1960, 1961) showed previously that thc reduction of lipoyl derivatives by DPNH catalyzed by thc pig heart dihydrolipoic dehydrogcnase is absolutely dependent 011 lhc presence of DPN. The DPN LLPpnrcntly prevents the coiivcmion of the cnzymc by excess DPNH to a c*ntalytically inuctive form in which thc flaviii is fully reduced, i.e. FADH2 - 15, (SH),. The 13’. coli dihydrolipoic dchydrogcnasc is inhihitd by prcincnhation with both 1)PNII and arsenite, h i t not by either substance alone. IIithiols wcrc more dfcctive than monothiols in reversing the inhibition. ‘l’hrse obscrvatioiis suggest that thc 13. coEi enzyme also contains a reactive disulfidr group. Finally, thc latter enzyme, as obtained from the pyruvate dohydrogenation cwriplcx by urea resolution apparently contains 2 molecwles of F’AI) per mole of enzyme ( h i k e , Carroll, arid Reed, iinpublishcd rcsults). b’urt her. understanding of the implications of this finding could conc*eivahlyrcquirc modification of the proposed reaction sequence [Eqs. (10 ll)]. 5 . Functional Form of Lipoic Acid Early studies on the distribution of lipoic acid in tissues indicatcd that it was tightly bound to protein. Thus, it was not extractable by hot water or by lipid solvents h i t was released only by hydrolysib with acid, alkali, or crude proteolytic enzymes (Reed el al., 195lb; C h s a l u s et al., 1952; Pat,t,ersonet al., I!K4). The bound lipoic acid in purificd preparations of the pyruvate :tnd a-kctoglutaratc dehydrogenation cbomplexes wits riot released I)y extrartion with hot alrohol-cthrr or with hot trichloroarctic acid (Koikc ct al., 1960a; Snnadi ct al., 195%). Thcsc latter observations provided a furthrr indiwtion that the lipoic mid is bound covalently to protein. Elucldation of the naturc of the linkage of lipoic acid to proteiii was csscntial for verification of the postulated reactions (7) through (11) and for further

BIOCHEMISTRY OF LIPOIC ACID

25

clarification of mechanism. As will be seen below, studies of component,s and conditions necessary for incorporation of lipoic acid into the apopyruvate dehydrogenation system of X. faecalis and for release of bound lipoic acid from the E. coli pyruvate and a-ketoglutarate dehydrogenation complexes indicated t,hat in it,s functional form lipoic acid is bound to protein in covalent linkage through its carboxyl group. Subsequently, a direct approach involving degradation of the E. coli complexes containing bound radioactive lipoic acid revealed that t,he lipoyl moiety is linked to the Eamino group of a lysine residue. Reed and co-workers (Leach et al., 1955; Iiecd et al., 1958a) observed that, preincubation of extracts of lipoic acid-deficient, S. faecalis cells with lipoic acid gave preparations which were capable of catalyzing the oxidation of pyruvate or a-ketobutyrate. Experimerits with S36-labcled lipoic acid showed that, during the preincubation lipoio acid was converted to a protein-bound form. Fractionation of the lipoic acid-deficient extracts revealed requirements for ATP and two protein fractions for incorporation of lipoic acid into the apopyruvate dehydrogenation system. One of the essential fract,ions produced lipohydroxamate and pyrophosphate when incubated wit,h lipoic acid, ATP, and hydroxylamine. Lipoic acid and ATP, but neither of the two prot,ein fractions, were replaceable by synthetic lipoyl adenylate. The reaction sequence (28) t,hrough (30) was proposed (Reed, 1958) to explain these results. Reaction (28) is visualized as an activation of the carboxyl group of lipoic acid through formation of lipoyl adenylate, which

+ ATP + lipoic acid El-lipoyl-AMP + EII lipoyl-En + APDS

EI

+ PP lipoyl-EI1 + AMP + EI lipoyl-APDS + EII EI-lipoyl-AMP

4

(28)

4

(29)

4

(30)

appears to be bound to a lipoic acid-activating enzyme (El). Reactions (29) and (30) are visualized as a t,riLnsfer of the lipoyl moiety to the apopyruvate dehydrogenation system (APDS). These lat,ter two reactions are apparently cat,alyzed by a t,ransfer enzyme of low molecular weight, (Lippmann and Reed, unpublished resuks) . A lipoic ac:id-incorporating system has also hcen det,ected in and isolated from extracts of E. coli (Reed el al., 1958a), suggeshg a general significance for t,he enzyme syst,em. Attcmpts to sepurat,e the If. coli system into two components have been unsuccessful. Support for the proposal that lipoic acid is bound in the pyruvate dehydrogenation complex in covalent linkage through its carboxyl group was furnished by studies with a hydrolytic enzyme, “lipoyl-X hydrolase,” obtained from ext,racts of 8. faecalis (Reed et al., 1958b). Incubation of the E. coli pyruvate and a-ketoglut’arate dehydrogenat,ion complexes with lipoyl-X hydrolase released approximately 96 % of the hound lipoic acid (Koike and Reed, 1960) and resulted in a loss of the DPK-linked a-ket,o

26

LE8TER J. REED

acid dehydrogenation activity [Eq. (3)]. The inactive complexes were separated from the hydrolase by sedimentation of the former in an iiltracentrifuge. The pellets were incubated with radioactive lipoic acid, ATP, and the lipoic acid-activating system. The complexes were separated froin the activating system by ultracentrifugation and were found to contain approximately as much bourid lipoic acid as the untreated complexes arid to exhibit essentially the same enzymatic activity. When E . coli is grown aerobically in the presence of lipoic a ~ i d - the S~~~ latter substance is incorporated into the pyriivate and a-kctoghitarate dehydrogenation complexes (Nawa rt al., 1060; Koike et al., 1960a). These radioactive complexes have been isolated in a highly purified state and used to rlucidatc the nature of the moiety to which lipoic acid is bound. Both radioactive romplexes were' oxidized with performic acid and then p:Lrt idly hydrolyzed with 12 N hydrochloric acid (3 hours at lOSOC;.). From the hydrolyzates thcrc was isolated in good yield a ninhydrin-posit ivc, radio:ictive conjugate which was identified as N‘-(6,S-disulfooc.tanoyl)-r,-lysirie(XII) by degradation and synthesis (Nawa rt al., 1960). 0 ~ c H Z ) , C N H (IIC H z ) , C H C O 2 H HO,S*’

*SO,H (XII)

I NHZ

The amino acid sequence about t,he Nf-lipoyllysine residue in the two enzyme complexes has been determined (Daigo and Rced, 1962b). T h c sequence Gly Asp. c-Lipoyl-Lys. Ala is prcscnt in the pyruvatc dehydrogenation cornplcx arid the sequence Thr .Asp * e-Lipoyl-Lys .Val. (Va1,Leu) Glii is present in the a-ketoglutarate dehydrogenation complex. It is thus apparcnt that. both complexes contain the sequence Asp c-Iipoyl-Lys, but, ot,herwisc t)hcscqiwnccs arc different. This difference in amino acid sequencc is probably rcsponsihle, a t least, in part,, for the substrate specificity of the two complexes. The evidence prcsent,ed ahove demonstjrut,es that, in its functional form in t,hc E. coli pyruvate and a-kctoglutarat#e dehydrogenation complexes, lipoic acid is bound in amide linkage with a protein c-amino lysine group. That this linkage niay also be present in mammalian a-keto acid dehydrogenation complexes is suggested by the rcport of Seaman (1959) that a hydrolyt,ic enzyme from bakers’ yeast, which resembles lipoyl-X hydrolase, released hound lipoic acid from the former complexes. In the R . coli systems it appears that lipoyl-X hydrolase cleaves the covalent bond between lipoic acid and a protein +amino lysinc group. Rcactivation of the apoenzyme

lJIOCIIEMlSTRY OF LlPOIC ACID

27

presumably involves re-forming this bond and requires incubation with lipoic acid, ATP, and a lipoic acid-activating enzyme system. The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes, It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as N'-biotinyl-L-lysine (Wright et al., 1952; Peck et al., 1952). Biotin is now known to be the prost,hetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the €-amino group of a lysine residue. 6. Structural Organizatim of a-Keto Acid Dehydrogenation Complexes

Pyruvate and a-ketoglutarate dehydrogenation systems which catalyze over-all reaction (3) have been isolated from mammalian and bacterial cells as enzyme complexes of high molecular weight (see Section 111, A). Recent studies on t,hc composition, resolution, and reconstitution of these complexes have contributed significantly to further underst,anding of the mechanism of lipoic acid action arid have provided insight of possibly general significance with respect to the mechanism of interaction between bound prosthetic groups in enzyme complexes. It has been presumed that as many as four separate enzymes are involved in over-all reaction (3), since the latter reaction apparently occurs in a sequence of four steps, i.e., decarboxylation, acyl generation, acyl transfer, and electron transfer. However, previous attempts to separate the individual enzymes and thereby confirm t,he proposed reaction sequence have met with only limited success. No separation of the mammalian pyruvate and a-ketoglutarate dehydrogenation complexes could be obtained during purification (Jagannathan and Schweet, 1952; Schweet et al., 1952; Sanadi et al., 1952). However, both the pyruvate and a-ketoglutarate dehydrogenation systems of E. coli were easily separated into two fractions during purification-fractions A and B and A' and B, respectively (Korkes et al., 1951; Hager and Gunsalus, 1953). Although complete separation was not achieved, it was apparent that fractions A and A' were rich in the enzymes catalyzing the decarboxylation, acyl-generation and acyl-transfer reactions [Eqs. (7-9)] and that fraction B was rich in dihydrolipoic dehydrogenase. Recently, Massey (1960b) achieved a separation of the pig heart a-ketoglutarate dehydrogenation complex into the flavoprotein component, dihydrolipoic dehydrogenase, and a colorless fraction similar in function to E. coli fraction A', by fractionation on calcium phosphate gel-cellulose in the presence of 2.5 M urea. Neither fraction alone was capable of catalyzing

rcaction (3), hut this activity was reconstituted on mixing the two fractions. I t t w I and b i k e (1961) achieved a separation of the E. coli pyruvate dehydrogenation complex into dihydrolipoic dehydrogenasc and a colorless component of high molecular weight by u modification of Massey’s prowdurc. When iiiixcd, these two components reassociated to produce a unit which rwmbled the original complex in cornpositioii, c~iayrnaticactivities, and stdimciitution ch:Lr.ar’tCl.ihfi~~S. l‘hc K . coli a-ketoglutaratr dehydrogrnst ion complex was separated in a siinilur manner into dihydrolipoic dehydrogenasc and a colorless component, and reassociation of these two cwmponent s wah demonstrated. It appears that thc urea resolution procedure dissocaiatcs the flavoprotein (E3 in Fig. I ) from the complexes, lcavirig a large colorless component which, as can be seen below, is itself a complex of the enzymes (El and E2)catalyzing reactions (7) through (9). Koike and Reed (1961) observed that in the presence of 0.02 A1 ethanolairiinr (pH 9.0-9.5) the colorless component obtained from the E. coli pyruvatc dehydrogcn:itiori complex separated into two components pyruvic carboxylase (El) and lipoic reductasc-transacetylasc (PI2). The pyruvate dehydrogenation comples was also treated with ethanolamine to dissociate the carboxylase, leaving a complex of lipoic reductase-t ransacetylwie and dihydrolipoic dehydrogenase, which in turn was separated into its two components by fractionation in the presence of 4 df urea. All three componcnts, carboxylase, lipoic rcductase-transa ylase, and flavoprotein, were required to reconstitute the DPN-lin oxidation of pyriivate [ICq. ( 3 ) ] .When the thrcc comporicrits were mixed in a ratio of 1.3: 1.0:0.5 by might, which corresponded to the relative ratio of these components in thc original comples, a large unit was produced whose composition, enzymatic uctivitieh, and sedimentation characteristic‘s rcsembled those of thc original cmiplex. Other sedimcntalion studies indicated that the carboxylase arid the flavoprotcin did not combine with each other, but these two components did combine indeperidcntly of each other with the lipoic rediictase-transucctylasc fruction. ‘I’h latter observations are corisisterit with the results of the resolution experiments, i.e. the flavoprotein (EJ can be dissociated from the complex with urea, leaving a complex of El and EL,arid Lhe carboxylase can be dissociated from the complex with ethanola[nine, leaving a complex of F:2 and E,. It would appear that there are highly specific binding sites on lipoic reductase-trans:~cetylasefor the carboxylase and for the flavoprotein. The resolut ion and reconstitution experiments described above indicate that the E. coli pyruvate dehydrogenation complex is a highly organized multicnzymc unit. The number of molecules of cwhoxylase and of flavoprotein per molecule of complex have been calculated. These numbers are about 12 for thc carboxylase and ahout 6 for thc flavoprotein. Each mole-

BIOCHEMISTRY OF LIPOIC ACID

20

r enzymc apparently contains 2 molrcules of FAD (Koike, Carroll and Rerd, unpublished results). The complex as isolated is partially resolved with resprct to thiamine pyrophosphate. However, assuming one molecrilr of the latter coenzyme prr molecule of carboxylase, the ratio of TPP to FAD in the fully active complex would be 1:l. The ratio of bound lipoic acid to FAD in the complex is approximately 3: 1 (Koike et al., 196Oa). Thus, there appears to be an “excess” of bound lipoic acid. As mentioned previously, the lipoic acid in the complex is bound to the lipoic rcductase-transacetylase component, which is itself a large unit. It is not yet clear whether this component is a complex of two separate enzymesa lipoic reductase and a dihydrolipoic transacetylase-or whether both activities reside in the same enzyme. However, the high lipoic acid content of this component does suggest that it is an aggregate of a smaller subunit, the minimal molecular weight of which would be approximately 30,000. ThP available data do not yet permit a conclusion as to whether or not the pyruvatc dehydrogenation complex is built up of a unit complex or “monomcric complex” of enzymes. Furt hrr study of the lipoic reductase-transacrtylase component should shed light on this question. As yet the mammalian and bacterial a-ketoglutarate dehydrogenation complexes have heen separated into only two components-the flavoprotein (Ex) and what would appear to hr a complex of an a-ketoglutaric carboxylase (El) and a lipoic reductase-transsuccinylase (EJ. Thc pig heart a-ketoglutaratc dehydrogenation complex contains bound thiamine pyrophosphate, lipoic acid, and PAD in essentially a 1:1:1 ratio (Massey, 19CiOb). A minimal molecular weight of 260,000 has been calculated from these analyses. Since the molccular weight of the complex as isolated is approximately 2 million, it would appear that each molecule of thr latter is an aggregate of about 8 unit or “monomeric” complexes. The E. coli akctoglutaratr dehydrogenation complex also contains bound lipoic acid and PAD in a 1:1 ratio, and the unit molecular weight is approximately 240,000. The molecular weight of the isolated complex is approximately 2.4 million, suggesting that each molecule of the latter is composed of about 10 unit complexrs. This picture of the organization of the a-ketoglutarate drhydrogenation complexes may, however, be an oversimplification, in virw of t he apparent complexity of organization of the pyruvate dehydrogenation complex. The results of the resolution and rrconstitution experiments carrird out with the E. coli pyruvate dehydrogenation complex indicate that there are specific binding sites on the lipoic reductase-transacetylase component for the carboxylase and the flavoprotein. In other words, the latter two enzymes appear to be specifically oriented with respect to the lipoic acid bound to the lipoic reductase-transacetylase component. It is evident from the re-

30

LESTER J. NEED

action scqaenre (8) through (10) that the l~ouridlipoic arid undergoes a cyclical series of transformations, i.p. reductive acylation, acyl transfcr, and reoxidation. These transformations are illustrated schematically in Fig. 5 . It is to be notcd lhut the lipoic acid which is bound to one enzyme niust interact with “acetaldehyde-thiamine pyrophosphate” which is bound to a second enzyme (carboxylase) and also with the reactive disulfide group DPN

H+

co2

transocetylase

Corboxylose

Ac-SCaA

Pyruvate

‘

CoASH

FIG.5. Schcmatic representation of the central role which bound lipoic acid plays in the oxidative decarboxylation of pyruvatc.

_II-

1

4

A

.

I p

~

5-s

PIC;. 6. Functional form o f lipoic acid in Bscherichia coli pyruvate and a-keto-

glutjarate dehydrogenation complexes. The carboxyl group of lipoic acid is botlnd in aniide linkage t o the €-amino group of a IyNine residue, providing a flexible arm of npproximntely 14 A f o r the rcactive dithiolane ring.

of a third enzyme. Thcsc interactions between prosthetic groups of separate enzymes occur within a complex in which there is apparcntly restricted movement, of the individual enzymcs and no dissociation of intermediates. A possible solution to this cnigma is provided by the discovery of Reed and vo-workers (Xawa et al., 19tiO) that lipoir acid is bound to the t-amino group of a lysinc residue. This attachment provides a flexible arm, approximately 14 A in length, for thc reactive dithiolane ring of lipoic acid (Fig. 6), conceivably permitting movement of the latter between the bound “acetalde-

BIOCHEMISTRY OF LIPOIC ACID

31

hyde-thiamine pyrophosphate” of the carboxylase, the site for acyl transfer to CoA, and the reactive disulfide of the flavoprotein (Fig. 7). A similar conclusion was arrived a t independently by Green, Bock, and Criddle (cf. Green and Oda, 1961). It is also conceivable that the “excess” bound lipoic acid in the pyruvate dehydrogenation complex permits a wider excursion between the “acetaldehyde-TPP” of the carboxylasc and the reactive disulfide of the flavoprotein. I n other words, the distance between the “acetaldehyde-TPP” and the reactive disulfide may be too large to be encompassed by the excursion of a single lipoyllysyl moiety, thereby necessitating interaction between two or three such moieties (Fig. 8). Such

I n

CoASH

Ac-SCoA

FIG.7. A schematic representation of the possible rotation of a lipoyllysyl moiety between “aldehyde-thiamine pyrophosphate” bound t o carboxylase (C), the site for acyl transfer t o CoA, and the reactive disulfide of t h e flavoprotein (F). The lipoyllysyl moiety is an integral part of lipoic reductase-transacetylase (LRT).

interaction is visualized as comprising thiol-disulfide interchange and acyl transfer (cf. Fig. 3). Such interaction between bound lipoyl moieties is presumably not necessary in the smaller a-ketoglutarate dehydrogenation complex. The 1:l:l ratio of bound TPP, lipoic acid, and FAD in the latter complex suggests that the excursion of a single lipoyllysyl moiety can aecoinmodate one “succinaldehyde-TPP” and one prosthetic disulfide group (Fig. 7).

B. OTHERPOSSIBLE ROLESOF LIPOICACID Effects of lipoic acid and its derivatives have been noted with a variety of biological systems (see reviews by Reed, 1957, 1960). Since the site of action of lipoic acid in these systems is either unknown or poorly defined, the present discussion will be limited to general comments on other possible

:z'2

LESTER J. REED

biological rolcs of lipoic acid. It would appear that a majority of the rcported cffccts are due iiol to t~ vitamin-like furiction of lipoic acid, but rather to thiol-disulfidc inttwhangr rractions with prolciri disulfide or t hiol groups, or to inhibition of rnctal-containing cneymt's by dihydrolipoiv arid. It is pert iricrit to note that thc rate of thiol-disulfide intcrchange reactions can VLLIYmarkedly with the nature of the disulfide and thiol involved (see rcview by Boyer, 19.59). Thus trimethylene disulfide (1,2-dithiolane) is cleaved a t a fastcr rate than n-butyl disulfide by n-butane thiol, and the

I

n

CoASH

Ac

-

SCoA

FI~:.8. A scheinntic representation of possible interactions between several lipoyllysyl nioiet ies i n the E . coli pyruvate dehydrogenation complex. These inter:tctions may involve thiol-dihulfide interchange and acetyl tr:msfer. The arrowb describe the urea covered by cach lipoyllysyl moiety.

rate of cleavage of trimethylcnc disulfide by n-butanc thiol is much more rapid than by methyl thioglycolatr (Fava et aE., 19357). Also, it has bccn obscrved frequciitly that thiols differ in their ability to reduce protein disiilfidc linkages or to activate certain cnaymes. E'or example, thioglycolate is ahoiIt fifty times as cfrectivc as vysteine in reducing thc lactogenic hornic)ric (k'racnkcl-Conrat et al., 1942). 'l'hcse arc clearly rate, not cyuilibrium, phenomena since thc reduction potentials for thc cystcinc, thioglycolate, and dihydrolipoate systems are nearly equal, i.e., about -0.32 volts a t pH 7.0 (Iiolthofi. et al., 1955; Tariaks et al., 1955; Ke, 19.57). It seems possible thereforc that what may appear to be a specific stimulation (or inhibition)

BIOCHEMISTRY OF LIPOIC ACID

33

of an enzyme or enzyme system by lipoic acid or its derivatives is merely a manifestation of the faster rate of oxidation or reduction of protein sulfhydryl or disulfide groups, i.e., thiol-disulfide interchange, by the lipoic-dihydrolipoic system than by other disulfides or thiols. At present there is no convincing evidence of a natural function of lipoic acid other than its established role in the CoA- and DPN-linked oxidative decarboxylation of a-keto acids. E’urt,hermore, there has been insufficient, recognition of the difficuky of obtaining such evidence. Undue emphasis has been placed on inhibition by arsenit,e and reversal by BAL, but not by monothiols, as n criterion of lipoic acid participation in a n enzymatic process. It is becoming increasingly apparent that enzymes which do not contain lipoic acid also exhibit this behavior. Two such examples are a Pseudomonas aldehyde dehydrogenase (Jakoby , 1958) and dihydrolipoic dehydrogenase (Section 111, A, 4). In these latter cases it appears that arsenite is inhibitory by virtue of combination with vicinal protein sulfhydryl groups, prcsumably those of cystcine residues. It is suggested therefore that unequivocal evidence tjhat lipoic acid is an integral part of an enzyme should include a demonstration of inactivation and reactivation of the enzyme accompanying, respectively, release and reincorporation of lipoic acid (see Section 111, A, 5). Calvin and co-workers (Calvin, 1954; Barltrop et al., 1954) speculated that lipoic acid plays a key role in photosynthesis as a compound involved in the primary conversion into chemical energy of the light quanta absorbed by chlorophyll. They visualized a transfer of electrons from the chlorophyll aggregate to lipoic acid to produce dihydrolipoic acid, which in turn reduced DPN or T P N (Bassham and Calvin, 1956). Although Calvin’s hypothesis is at*t,ractiveand has st,imulst,edresearch in this field, there is as yetj no unequivocal evidence t,o support it. Moreover, the finding of Arnon et al. (1959) that phot,oreduction of TPN accompanied by oxygen evolution and phosphorylation in a chloroplast system is not inhibited by 2 x M arsenite appears to render unlikely t,he participation of lipoic acid, a t least in its dithiol form, in the light reactions of photosynthesis. The novel requirement of lipoic acid for growth of Butyribacterium rettgeri on a medium containing lactate as the main energy source, but not on a medium containing glucose or pyruvate (Barker, 1954) has been investigated further by Wittenberger (1961). The possibility that lipoic acid functions in the transport of lactate into the cell appears to have been ruled out. Evidence was adduced for a role of lipoic acid as a carrier of electrons originating in lactate. However, it appears that further definition of this system will be necessary before the precise role of lipoic acid can be specified.

34

LESTER J. REED

IV. CONCLUDING REMARKS There is coiisiderable evidence that in bacterial and animal cells, arid presumably in plant cells as well, lipoic acid is a prosthetic group of multienzyme complexes which generate acetyl CoA and succinyl CoA from pyruvate and a-ketoglutarate, respectively. The covalently bound lipoic acid undergoes a cycle of reductive acylation, acyl transfer, and reoxidation. These t’ransformations involve interaction between prosthetie groups of separat>eenzymes within the complexes. It has been demonstrated in the (:useof the E. coli pyruvate and a-ketoglutJaratedehydrogenation complexes t,hat lipoic acid is bound in amide linkage to the e-amino group of a lysine residue. It is suggested that this attachment provides a flexible arm which permits of successive collisions of the dithiolane ring with other functional groups in the complexes. Further irivcstigatiori of the st,ruetural organization of Lhe complexes holds promise as an intriguing and rewarding area of research. Although thew is a t present, no convincing evidence of a natural function of lipoic acid ot,her than that, of a prosthetic group i n a-ket,o acid dehydrogcnation complexes, it is conccivahle tJhatjlipoic acid may function ill ot,her cmynie systems as an acyl or electron carrier. The unique chemical properties of the dithiolane ring in lipoic acid and t,hc vicirial dit>hiolgroups in dihydrolipoic acid suggest the possibility that these (:ompounds could exert phaririacological efY s through thiol-disulfide int,crchange reactions with protJeindisulfide and thiol groups or tJhroughinhi bit,ion of met,al-containing enzymes. This possibility merits further considcration in fuLure research. Synthesis of analogs of lipoic acid with the object of obtaining an effective antagonist should be encouraged. The availability of an effective antagonist would permit invest,igat)ionof the biological role of lipoic acid in diverse orgaiiisms arid possibly enable a lipoic acid deficiency to be cstablished in animal species. 3-Acetylthiamine pyrophosphate appears to be yet another form of “active acetate.” It has been assigned a key role in the lipoic acid-linked oxidative decarboxylation of pyruvate as the primary product of the oxidation of “active acct,aldehyde,” i.e., 2-hydroxyethylthiamine pyrophosphate. It has been proposed that heetylthiamine pyrophosphate is an intermediate in all oxidative transformations of pyruvate and that 2-succinylthiamine pyrophosphate plays a similar role in oxidation of a-ketoglutarate. Further evaluation of this proposal is anticipated in the near future. REFERENCES Arrioii, L). I., Whatley, F. R., and Allen, M. B. 1959. Biochim. et Biophys. Acta 32, 47-57.

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