ANALYTICAL

BIOCHEMISTRY

192,49-54

(1991)

An Acyl-Coenzyme A Chain Length Dependent Assay for 3-Oxoacyl-Coenzyme A Thiolases Employing Acetyldithio-Coenzyme A’ Louise V. Wrensford, Chris Coppola, and Vernon E. Anderson3 Department of Chemistry, Brown University, Providence, RhodeIsland 02912

Received

March

6, 1990

0 An assay for 3-oxoacyl-coenzyme A (3-oxoacyl-CoA) thiolases is described. The reaction utilizes acetyldithio-CoA as the nucleophile and variable chain length saturated acyl-CoA’s as the electrophiles. The properties of the 3-oxoacyl-CoA dithioester product, notably a pKe of 6.6 f 0.1 and an extinction coefficient of 2 1,600 cm-’ M-’ for the enethiolate at 357 nm, make it possible to spectrophotometrically follow the reaction in the thermodynamically unfavorable carbon-carbon bondforming direction. These properties eliminate both the background decomposition and the dependence on Mgx+, chain length, and pH that complicate assays with 3-oxoacyl-CoA substrates. Purified thiolase I from pig liver was 140-fold more active with butyryl-CoA as the electrophile than with acetyl-CoA and 38-fold more reactive with hexanoyl-CoA than with myristoyl-CoA. Beef liver homogenate showed a much greater relative activity with myristoyl-CoA as the electrophile than either purified pig heart thiolase I or pig heart homogenate. The analysis of the separation of thiolases by anion-exchange chromatography is simplified and further suggests the existence of isozymes with varying chain length specificities. o 1991 Academic PWS, I~C.

Thiolases (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) catalyze the reversible Claisen condensation of an acyl-CoA with acetyl-CoA (AC-COA).~ As shown in Eq. [ 11,

’ Supported in part by Grant GM36562 from the tutes of Health. ’ Recipient of NIH minority predoctoral fellowship. a To whom correspondence should be addressed. ’ Abbreviations used: AcAc-CoA, acetoacetyl-coenzyme acetyl-coenzyme A, Ac(=S)CoA, acetyldithio-coenzyme dithiothreitol. 0003-2697/91 Copyright All rights

$3.00 0 1991 by Academic Press, of reproduction in any form

National

Insti-

A; AC-CoA, A; DTT,

R

+ thiolase-Cys-S

A

p

SCoA

0 R

+ CoA

A

PI

S-Cys-thiolase

the acyl-CoA is initially transferred to an active site cysteine thiol (2,lO). The a-carbon of AC-CoA reacts as the nucleophile in an acyl substitution reaction forming the 3-oxoacyl-CoA product (Eq. 2).

0 A

R

0 + S-Cys-thiolase

A

SCoA = + thiolase-Cys-S

[2]

SCoA There are at least four different thiolases present in most mammalian tissues (3,4). Two, one mitochondrial and one cytoplasmic, are specific for AC-CoA in Eq. [l] or acetoacetyl-CoA (AcAc-CoA) in the reverse reaction of Eq. [2]. These enzymes are involved in ketone body metabolism and isoprenoid biosynthesis. A peroxisomal thiolase capable of cleaving long chain 3-oxoacyl-CoA’s has more recently been identified (5). A single thiolase, referred to as thiolase I, lacking rigid chain length specificity is isolated from mitochondria (1) that functions in the @-oxidation of fatty acids. There is experimental evidence suggesting that there may be additional mitochondrial thiolase isozymes demonstrating varying chain length specificities. Middleton observed two activity peaks on isoelectric focusing, both having activity with 3-oxohexanoyl-CoA (3). Of the other three enzyme activities in P-oxidation, long-, medium-, and short49

Inc. resewed.

50

WRENSFORD,

COPPOLA,

chain acyl-CoA dehydrogenases have been identified (6), and long-, and short-chain enoyl-CoA hydratase activities have been separated from pig heart (7). Analogy would suggest that thiolase isozymes exhibiting varying chain length specificities may be identified as well. The current assay for thiolase chain length specificity is complicated by four features. (A) The 3-oxoacyl-CoA substrates are not readily available. The 3-0~0 acids or derivatives are not readily available for thioesterification with chain lengths beyond C, and, although the method is robust, above C, the 2-alkynoic acids are not available for the synthesis of 3-oxoacyl-CoA thioesters via the crotonase-catalyzed hydration of 2-alkynoylCoA thioesters (8). The “routine” synthesis remains enzymatic P-oxidation of the saturated (9) or p-unsaturated acyl-CoAs (1). (B) The extinction coefficient of the 3-oxoacyl-CoA is a function of chain length, substrate concentration (l), pH, and Mg2+ concentration (10). (C) The 3-oxoacyl-CoAs are unstable in their ionized form, decomposing through a ketene elimination (11). This results in a large background for low activity assays. (D) The apparent K,,, for the 3-oxoacyl-CoA has to be corrected for the fraction present as the Mg2+ enolate (10). These problems could be overcome if the assay could be determined in the C-C bond-forming direction. Although possible for the dimerization of AC-CoA, the measured rate in the presence of acyl-CoA and AC-CoA will always have a component due to the dimerization of AC-CoA. Furthermore, the equilibrium constant for formation of the 3-oxoacyl-CoA is extremely small, 1 X 10e5 (lo), and the reaction must be coupled to the 3-hydroxyacyl-CoA dehydrogenase-catalyzed oxidation of NADH. The recent demonstration that acetyldithioCoA (Ac( =S)CoA) will not acylate the active site thiol of the biosynthetic thiolase from Zoogloea ramigera, but will readily serve as the nucleophile in the C-C bond-forming condensation (12) prompted us to determine whether this was true of mitochondrial thiolases as well. The product of the condensation reaction (Eq. [2]) is the ene(thio)late of the 3-oxoacyldithio-CoA. The pK, of 6.6 for 3-oxoacyldithio-CoAs, the sixfold increase in the equilibrium constant for carbon-carbon bond formation (12) and the large extinction coefficient make it possible to use Ac(=S)CoA with varying chain length acyl-CoAs in a thiolase assay demonstrating chain length specificity that obviates the four difficulties of the current assay.

MATERIALS

AND

METHODS

Materials. AcAc-CoA was synthesized as described previously (9). CoA (Li+ salt), acyl-CoAs and all other biochemicals were obtained from Sigma. Lawesson’s re-

AND

ANDERSON

agent and thiophenol were from Aldrich. Isolation and purification of all reaction products were carried out by HPLC on a Perkin-Elmer Series 400 liquid chromatograph using an Econosphere (Alltech) C,, reverse-phase column (10 X 250 mm) operating at a flow rate of 2 ml/min. ThioZuse. Purified pig heart thiolase was a generous gift of Dr. H. Gilbert of Baylor Medical College. Enzyme activity was determined by the standard assay monitoring the thiolysis of AcAc-CoA (lo), one unit being the disappearance of 1.0 pmol of AcAc-CoA min-‘. Pig hearts and beef livers were obtained fresh from local slaughter houses and stored frozen at -2O’C. For kinetic studies on homogenates, 10 g of beef liver and 30 ml of homogenization buffer (20 mM Tris-HCl, pH 7.0, 0.1 mM EDTA) were homogenized in a Waring blender operated at high speed for 30 s. A l-ml portion of the crude homogenate was centrifuged for 30 min. in an Eppendorf microcentrifuge and assayed for thiolase activity and protein concentration. Pig heart homogenates were prepared as described by Staack et al. (1). All protein concentrations were determined by Bradford protein assay (Bio-Rad) with bovine serum albumin as the standard. Ac(=S)CoA. Dithioesters were synthesized as described by Wlassics and Anderson (13). 80 pmol of Sphenyl dithioacetate was added to a solution of 10 pmol of CoA (Li+ salt) in 4 ml of a 1:l (v/v) mixture of 0.1 M sodium phosphate buffer, pH 85:ethanol with ethyl acetate added dropwise until a homogeneous mixture was obtained. After being stirred for 20 min at room temperature, the mixture was acidified with a few drops of phosphoric acid to pH 4, and 5 ml of ethyl acetate added. The aqueous layer was then extracted twice with 2-ml aliquots of ethyl acetate. Residual ethyl acetate was removed under a stream of nitrogen and the Ac(=S)CoA titrated to pH 7 with solid Tris for use in enzyme assays and was used without further purification. The concentration of the solution was determined by the absorbance at 306 nm (c = 11,000 M-kmwl). Unreacted CoA was less than 3% of the stock Ac(=S)CoA solution as determined by assay for free thiols with 3-carboxy-4-nitrophenyl disulfide (14). The ratio of the absorbance at 259 nm to the absorbance at 306 nm was 1.31, indicating a 1:l ratio of adenine to dithioester. Decomposition products formed after storage over several days were removed by HPLC when this ratio exceeded 1.37. The column was preequilibrated with 20% MeOH in 25 mM NaH,PO, and eluted with 20% MeOH in water. The fractions containing Ac( =S)CoA may be pooled, taken to dryness under reduced pressure and stored desiccated at -20°C with little decomposition. 3-oxohexanoyldithio-CoA 3-Oxoacyldithio-CoA’s. and 3-oxooctanoyldithio-CoA were produced in 100~pg

VARIABLE

240

280 320 WAVELENGTH (nml

360

CHAIN

LENGTH

400

FIG. 1. Ultraviolet spectra of 3-oxohexanoyldithio-CoA. The 3-0xohexanoyldithio-CoA was synthesized from S-butyrylthiocholine and Ac( =S)CoA as described under Materials and Methods. The spectra were taken in NaH,PO,, pH 5.0 (a); and Tris-HCl, pH 8.0 (b).

quantities using S-butyryltbiocholine and 3-oxooctanoyl-CoA as acyl donors. 3-Oxooctanoyl-CoA was prepared from 2octynoyl-CoA by the method of Thorpe (8). To obtain the 3-oxoacyldithio-CoAs, 300 pM Ac( =S)CoA and either 8 mM S-butyrylthiocholine chloride or 300 pM 3-oxooctanoyl-CoA were incubated with 0.013 unit of thiolase in 1.0 ml of 0.1 M 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, pH 9.0, at ambient temperature. When there was no further increase in absorbance at 357 nm, the mixture was acidified with 25 ~1 of acetic acid. Product identification. The acidified reaction mixture was purified by HPLC. The solvent program for purification of the C, product consisted of a lo-min isocratic step of 25/50/25 (v/v/v) MeOH/H,O/O.l M NaH,PO, followed by an 18-min linear gradient to 50/ 25125 MeOH/H,O/O.l M NaH,PO, and held until product eluted at 40 min. For the C, product the isocratic step was 10 min at 45/15/25 followed by a 12-min linear gradient to 60115125. The product eluted at 40 min. Fractions containing the 3-oxoacyldithioesters were concentrated under vacuum. Ultraviolet spectra of the 3-0~0 dithioesters were taken at pH 5.0, 0.1 M sodium phosphate, and pH 8.0,0.08 M TrisHCl. The absorbance ratios A,, (pH 8.0)/A,,g (pH 5) of the C, and C!, products were found to be 1.40. Assuming the absorption at 259 nm is due to the adenine, tZb9 = 15,400 M-’ cm-l, the es57 is 21,600 M-‘Cm-’ and is independent of chain length. Z’hiotase assay. The reaction was monitored by the increase in absorbance at 357 nm due to the formation of the enolate of the 3-oxoacyldithio-CoA product (es67 = 21,600 M-’ cm-‘). The assay mixture contained 85 mM Tris-HCl, pH 8.5, at 25“C, 200 j&M Ac(=S)CoA, and varying concentrations of fatty acyl-CoA esters in a final volume of 1 ml. The reaction was started by the

THIOLASE

51

ASSAY

addition of the thiolase after equilibration of 25°C for 3 min. The concentration of the stock acyl-CoA solutions were determined using cZ9 of 15,400 M-’ Cm-‘. Concentration ranges for the fatty acyl CoA esters were: C,, 30 ~~-1.1 mM; C,, 41 ~~-1.6 mM; C,, 23-286 PM; C,, 24151 KM; cIo, 5-80 PM; cm, 14-109 PM. Initial velocities were determined manually from the initial slopes of plots of ASS7 versus time. The V,, and K, values for AC-CoA, which displayed substrate inhibition, are the maximum observed rate and the interpolated substrate concentration that results in half of the experimental maximum velocity. These apparent kinetic parameters systematically underestimate the true V,,, and K,,, values, but are the useful parameters when considering an assay to differentiate between thiolases that use just AC-CoA to acylate the active site and thiolases of broader specificity. The apparent V,,,, and K, values for all longer chain acyl-CoA’s were determined by a nonlinear least-squares fit (u/E,)

= V,,[(acyl-CoA]/(K,

+ [acyl-CoA]).

[3]

For the beef liver homogenate only V,,, was determined by using acyl-CoA concentrations of two and three times the K’, determined for the purified pig liver thiolase I. Fractogel anion-exchange chromatography of beef liver homogenate. Beef liver (25 g) was minced and added to 75 ml of 25 mM Tris-HCl, pH 7.1, containing 1 mM DTT, 1 mM EDTA, and 5% v/v glycerol. The mixture was homogenized in a blender at top speed for 2 min and centrifuged at 15,000g for 1 h. A lo-ml aliquot of the supernatant containing 35 mg of protein/ml was applied to a 1 X 15 cm trimethylaminoethyl Fractogel column

L 254

282

310

338

366

WAVELENGTH

FIG. 2. Spectrophotometric titration of 3-oxooctanoyldithio-CoA. The 3-oxooctanoyldithio-CoA was synthesized from 3-oxooctanoylCoA and Ac(=S)CoA as described under Materials and Methods. The buffers used from lowest to highest absorbance at 357 nm were sodium phosphate, pH 5.0, 3-(N-morpholino)propanesulfonic acid, pH 6.0 and pH 7.0, Tris-HCI, pH 7.4, pH 7.6, pH 7.7, and pH 8.0, respectively.

52

WRENSFORD,

COPPOLA,

AND

TABLE

Kinetic

Constants Purifled

the Thiolase

thiolase

Km (app) (CIM)

Acyl-CoA”

of

1

Reaction

with Substrates

I

Pig heart K,

V,, bw) (units/mg)

21’ 80r 12 58 zk 3 25 k14

ANDERSON

c2* c4 C6 C8 Cl0 Cl2 Cl4

o-094* 13 + .5 3.6 f .l 4.4 + .8

6.0 f .8 6.5 f 1

0.25 k .02 0.094 f .Ol’

AcAcCoA’

3.5 f .3

10.1 * .2

of Varying

homogenate

(apP) (PM)

52.6’ 99.1 +199.2 + 24.9 k 10.7 + NA NA

Chain Length

Beef liver homogenate VW bpp) (units/mg)

V,, (m-4 (units/mg) 0.00336 0.14 + .Ol 0.961 + .005 0.047 f .OOl 0.017 +- .OOl

An acyl-coenzyme A chain length dependent assay for 3-oxoacyl-coenzyme A thiolases employing acetyldithio-coenzyme A.

An assay for 3-oxoacyl-coenzyme A (3-oxoacyl-CoA) thiolases is described. The reaction utilizes acetyldithio-CoA as the nucleophile and variable chain...
640KB Sizes 0 Downloads 0 Views