human liver long-chain 3-hydroxyacyl-coenzyme a dehydrogenase is a multifunctional membrane-bound...

Vol. 183, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 16, 1992 Pages 443-448

HUMAN LIVER LONG-CHAIN 3-HYDROXYACYL-COENZYME A

DEHYDROGENASE IS A MULTIFUNCTIONAL MEMBRANE-BOUND BETA-

OXIDATION ENZYME OF MITOCHONDRIA

Karen Carpenter’, Rodney J. Pollitt2 and Bruce MiddletonI*

IDepartment of Biochemistry, University of Nottingham Medical School, Queen’s Medical Centre,

Nottingham NG7 2UI-L England, U.K.

2Department of Paediatrics, Childrens Hospital, Sheffield SlO 2’I’H, England, U.K.

Received January 2, 1992

SUMMARY: We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71kDa and 47kDa within a native stmcture of 230kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of Cl0 to Cl6 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl- CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with Gj and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane- bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl- CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation. 0 1992 Acadrmlc Press, Inc.

An inherited defect of mitochondrial fatty acid P-oxidation has been shown to be caused by

deficiency of a long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) [ 11. In some cases of

LCHAD deficiency [2,3] there is a parallel reduction in activity of long-chain specific 2-enoyl-

CoA hydra&e and 3-ketoacyl-CoA thiolase suggesting a molecular link between these long-chain specific enzymes of P-oxidation. We have shown [4] that human infant liver, like other mammalian

tissues [5], contains 2 mitochondrial HADs specific for short- and long-chain substrates; the

LCHAD accounts for up to 67+7% of total liver activity towards long-chain substrate, it shows no

activity with acetoacetyl-CoA and is a membrane protein of mitochondria. This paper describes

*To whom correspondence should be addressed.

Abbreviations: LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; SCHAD, short-chain 3- hydroxyacyl-CoA dehydrogenase; EDTA, ethylenene diamine tetraacetic acid; DEAE-C, diethylamino ethyl-cellulose; D’IT, dithiothreitol; SDS PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; CAPS, 3- [cyclohexylamino]-1-propanesulphonic acid; ECH, 2-enoyl-CoA hydratase; KAT, 3-ketoacyl-CoA thiolase; MD, malate dehydrogenase; CS, citrate synthase; MFE, multifunctional enzyme.


studies on the LCHAD purified from human infant liver and shows that it is unique among enzymes of mitochondrial P-oxidation in having multienzyme activities in a structure containing

non-identical, membrane-associated, subunits.

METHODS

Non-diseased, chromosomally normal, samples of premature and infant liver were obtained immediately after hospital post mortem. The age range was from 24 weeks gestation to 5 years. The interval between death and freezing in liquid N2 was E-48h. Synthesis of acetoacetyl-CoA (CJ) and assay of 3-hydroxacyl-CoA dehydrogenase with 3-ketoacyl-CoA substrates of varying chain length were as previously described [5]. Longer chain 3-ketoacyl-CoA (C&16) were prepared by the method of Thorpe [6]. Trans-3-enoyl-CoA and trans-2-enoyl-CoA were prepared from the free acids and CoASH by the mixed anhydtide method [7]. A3,A2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase were assayed with the appropriate acyl-CoA substrates as described in [El and [9] respectively. Acetyl-CoA formation from 2-enoyl-CoA compounds was assayed as previously described [lo].

To purify LCHAD, frozen livers were homogenised in 1OmM Tris-HCl pH 8.0 containing 1mM EDTA and O.OlmM leupeptin, membranes were then isolated, washed, solubilized in 40mM octylglucoside and chromatographed on DEAE-C as in [4]. After concentration by vacuum dialysis, the eluate was applied to a Hiload 26120 Superdex 200 column run on a Pharmacia Fast Protein Liquid Chromatography apparatus and eluted with 20mM Bis-tris propane-HCl pH 9.5 containing 1mM DlT, 1mM EDTA, O.OlmM leupeptin and 20mM octylglucoside. The fractions with highest specific activity with Cl6 substrate were applied directly to a Mono-Q HR 5/5 column and eluted with a 0- 1M NaCl gradient in the same buffer. After buffer exchange into 20&i phosphate pH 6.5 containing 0.5mM D’IT, 1mM EDTA, O.OlmM leupeptin and 20mM octylglucoside, the enzyme was applied to a Mono-S HR S/5 column and eluted with a gradient of O-1M NaCI. Protein bands on SDS PAGE gels were visual&d by silver staining. Electroblotting to PVDF membranes was carried out in CAPS buffer pH 11 containing 10% methanol. Amino acid analysis and N terminal sequencing was performed on Applied Biosystems Models 420H and 473A respectively using bands of protein cut from the PVDF blots.

RESULTS AND DISCUSSION

LCHAD purified from human infant liver typically showed a specific activity ranging from

15-35pmol3-ketohexadecanoyl-CoA (C16) reduced/min/mg protein and also gave this maximum

activity with 3-ketodecanoyl- (Clo) and 3-ketotetradecanoyl-CoA (C14) but was inactive with

acetoacetyl-CoA (a). In the physiological direction using enzymically generated 3-

hydroxyoctanoylCoA (Cs) as substrate purified LCHAD gave activity of 3.2 -7.4pmol/min/mg but

was inactive with 3-hydroxybutyryl-CoA (Ct). We have purified the LCHAD from livers of 4

individuals and in each case the pure enzyme was composed of 2 non-identical subunits. Fig. 1

shows the SDS PAGE analysis of the protein during the different stages of a typical purification

starting from washed, detergent solubilized membranes with a specific activity of 0.41pmol3-

ketohexadecanoyl-CoA reduced/min/mg (lane 1) and ending with a MonoS eluate of specific

activity 16.3pmol/min/mg (lane 5). Two polypeptides (Fig. 1; A, 71kDa and B, 47kDa) were

observed as bands with the same relative intensity throughout the purification of the LCHAD.

Amino acid analysis and N terminal sequencing showed that polypeptides A and B were present in

equimolar amounts in the native enzyme. The presence of non-identical subunits in pure LCHAD

contrasts with the single 3 1kDa polypeptide of the short-chain preferring mitochondrial HAD

(SCHAD) which has been shown to be a 75kDa dimer of identical subunits [l 11. We find that

native LCHAD has a molecular weight of 230kDa (unpublished gel filtration measurements using

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Fractionno. 12 14 15 16 17 18 20 22

A-w-O---

x 80

+A

.z ‘Z Y 60

I ‘Z 4

40

a 20

36- ’

29 - 0

0 0 10 1

14 16 18 20 22 24

1 2 3 4 5 2 Fraction number

Fig. SDS PAGE analysis of the purification of LCHAD. Lanes 1-5 contained fractions from a typical purification from human liver: 1, solubilised membranes; 2, DEAE-C, 3, Superdex 200; 4, Mono-Q; 5, Mono-S. A and B denote positions of 7 1kDa and 47kDa polypeptides respectively. Positions of protein standards &Da) are shown.

Fig. 2. Coelution of trans-2-enoyl-CoA hydratase (0) and 3-ketoacyl-CoA thiolase (0) with 3-hydroxyacyl-CoA dehydrogenase (0) activity in fractions from a high resolution Mono-S cation exchange column and comparison with the SDS PAGE analysis of equal aliquots of each fraction for polypeptide components (A = 7lkDa, B = 47kDa). Activities are relative to those of fraction 16 and were: ECH, 2.88pmol2-octenoyl-CoA/min; KAT, 0.25pmol3-ketodecanoyl- CoA/min; LCHAD, l.l6pmol3-ketohexadecanoyl-CoA/mm.

Superose 12 HR IO/30 run in 2OmM octylglucoside and 150mM KCl, pH 7). This would suggest

a subunit composition of A2B2 for LCHAD.

Pure LCHAD of specific activity 16.3pmol3-ketohexadecanoyl-CoA reduced/min/mg also

showed activities of 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase with substrates of acyl-

chain length from Cg to C16. The 2-enoyl-CoA hydratase activity with 2-octenoyl-CoA was

42.6pmol/min/mg and the 3-ketoacyl-CoA thiolase activity with 3-ketodecanoyl-CoA was

2.&.tmol/min/mg. Neither activity was detectable with Q substrates. These enzyme activities are

thus distinct and different from any described previously. We also attempted to measure As&--

enoyl-CoA isomerase activity in the purified enzyme since it has been identified as a component

activity of the rat liver peroxisomal multifunctional enzyme [II] and a long chain A3,A2-enoyl-CoA

isomerase of similar native molecular weight to the LCHAD has been reported in rat liver

mitochondria [ 121. However we could detect only traces of A3,A2-enoyl-CoA isomerase activity

(co.03 pm01 tram-2-octenoyl-CoA produced/min/mg) in the purified LCHAD.

To confirm the coexistence of multiple enzyme activities within the same protein, we

assayed for 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase through the peak of purified

LCHAD eluted from a Mono-S cation exchange column. Fig. 2 shows that these activities coeluted

in this highly resolving system and paralleled the relative intensities of polypeptides A and B

isolated on SDS PAGE from the same column fractions.

In addition, we have demonstrated (Fig. 3) that pure LCHAD would work as a

multifunctional enzyme. Thus it alone catalysed the generation of NADH (Fig. 3A) and 3-ketoacyl-

445


A

g ” 4 d

0 3 -’

b I

1Omin 4

1Omin 0 4

A

Fig. 3. Identification of multifunctional activities associated with the purified LCHAD enzyme. 40pM trans-2-octenoyl-CoA and 1mM NAD were incubated in 5OmM Tris HCI, 5OmM KCI, 5Ottg./ml bovine serum albumin, pH 9.0 at 3oOC. Absorption changes were followed at 340nm (A) and 303nm (B). Addition at (1) to A and B was 1 .@g of purified LCHAD. Incubation B also contained 1mM pyruvate, 25mM MgC12 and lO@-nl lactate dehydrogenase and 1OOpg CoASH was added at (2).

Fig. 4. Formation of acetyl-CoA from trans-2-octenoyl-CoA catalysed by the multifunctional LCHAD. lOO@ml CoASH, 1mM NAD, 1OmM malate and lO@ml malate dehydrogenase were incubated with 40l.tM (A) or 80l.tM (B) trans-2-enoyl-CoA in 50mM Tris HCl, 50mM KCl, 5O@ml bovine serum albumin, pH 9.0 at 30cC and the absorption was followed at 34Onm. Addition at (1) was 1.8pg of purified LCHAD and the acetyl-CoA formed during the incubation was measured by the increase in absorption following addition of lOltg/ml citrate synthase at (2).

CoA (Fig. 3B) from NAD and trans-2-octenoyi-CoA (or trans-2-tetradecenoyl-CoA, data not

shown) indicating the cooperation of activities of hydratase and dehydrogenase:

ECH LCI-IAD trans-2-enoyl-CoA ZE 3-hydroxyacyl-CoA + NAD z 3-ketoacyl-CoA +NADH.

When CoASH was added after the reaction reached equilibrium, the 3-ketoacyl-CoA thiolase

activity of the multifunctional enzyme caused a rapid removal of the 3-ketoacyl-CoA, reversing the

absorption increase at 303nm (Fig. 3B):

KAT 3-ketoacyl-CoA + CoASH Z acetyl-CoA + acyl-CoA.

Furthermore, we could observe the operation of the whole multifunctional enzyme system

by detecting the formation of acetyl-CoA from tram-2-octenoyl-CoA in the presence of NAD and

CoASH only:

ECH LCHAD KAT tram-2-enoyl-CoA = 3-hydroxyacyl-CoA z 3-ketoacyl-CoA zz acetyl-CoA + acyl-CoA.

+NAD +CoASH

446


Fig. 4 shows the formation of acetyl-CoA by the above reaction sequence. It was detected at the

end of the reaction (in the presence of malate and malate dehydrogenase) by the production of a

further burst of NADH on addition of citrate synthase as below [ 101:

MD+CS NAD + malate + acetyl-CoA - citrate + CoASH + NADH.

The amount of acetyl-CoA generated at the end of the reaction was proportional to the amount of 2-

enoyl-CoA initially present (Fig. 4 A, B).

Multifunctional enzymes (MFE) were thought to be characteristic of non-mitochondrial p-

oxidation systems: the primary structures and catalytic properties of the MFEs of rat peroxisomes

[13, 14,8], Candida tropicalis peroxisomes [15, 161 and E. cofi [17, 181 have been described.

Very recently, a mfunctional P-oxidation enzyme has been isolated [ 191 from membranes of rat

liver mitochondria and this, together with our findings, shows that the above generalisation is not

valid. The multifunctional LCHAD that we have purified from human liver differs somewhat from

the non-mitochondrial systems. While the MFE of E. coli has non-identical subunits of 78kDa and

47kDa and these, like the multifunctional LCHAD reported here, are organised in a A2B2 structure

[20], the E. coli MFE differs from the multifunctional LCHAD in that it is not a membrane enzyme

and is active with short chain (C4) substrates. Rat peroxisomal MFE is very different from the

multifunctional LCHAD, being a soluble enzyme [ 131 with a single subunit per molecule [ 141 and

without any thiolase activity.

Previous workers have suggested that the enzymes of mitochondrial P-oxidation could be

organised (21,22) but the evidence for organisation has only been indirect. The most compelling

evidence has come from careful studies using sensitive and specific detection methods [23,24]

which have demonstrated absence of any 2-enoyl-, 3-hydroxyacyl- or 3-ketoacyl-CoA compounds

accumulating as true intermediates during the oxidation of long-chain acyl-CoA compounds in

intact mitochondria. Our studies can explain these findings by providing the first direct evidence

for organisation of 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-

CoA thiolase P-oxidation activities in human liver mitochondria as a multifunctional, membrane-

bound enzyme.

ACKNOWLEDGMENTS

This work is supported by the Medical Research Council of Great Britain. We also thank the Nottingham University Medical School Trust Fund for financial assistance with the “osts of sequencing and the Wellcome Trust for a travel grant. We are grateful to Professor D.M. ;umbull for unpublished details of his patients and for the gift of 2-tetradecenoyl-CoA. We thank Dr J.K. Hiltunen for his helpful advice and Professor T. Hashimoto for discussion about the multifunctional enzyme of rat liver mitochondria.

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human liver long-chain 3-hydroxyacyl-coenzyme a dehydrogenase is a multifunctional membrane-bound...

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