Tài liệu Báo cáo khoa học: Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD) K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein pptx

Engel1 1 Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland 2 Research Unit for Molecular Medic

dehydrogenase (MCAD)

K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

Linda P O’Reilly1,*, Brage S Andresen2 and Paul C Engel1

1 Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

2 Research Unit for Molecular Medicine, University Hospital, Skejby Sygehus, Aarhus, and Institute of Human Genetics, Aarhus University, Denmark

Keywords

active site; enzyme deficiency; medium

chain acyl-CoA dehydrogenase (MCAD);

point mutations; protein folding

Correspondence

P C Engel, Department of Biochemistry,

Conway Institute, University College Dublin,

Belfield, Dublin 4, Ireland

Fax: +353 12837211

Tel: +353 17166764

E-mail: Paul.Engel@ucd.ie

Website: http://www.ucd.ie/biochem/

Enzymes

Medium chain acyl-CoA dehydrogenase

(MCAD; EC 1.3.99.3); long chain acyl-CoA

dehydrogenase (LCAD; EC 1.3.99.13); short

chain acyl-CoA dehydrogenase (SCAD;

EC 1.3.99.2); glutaryl-CoA dehydrogenase

(GCD; EC 1.3.99.7); isovaleryl-CoA

dehydro-genase (IVD; EC 1.3.99.10); electron

trans-ferring protein (ETF; EC 1.5.5.1).

*Current address

Department of Molecular Genetics and

Biochemistry, University of Pittsburgh, 200

Lothrop Street, Pittsburgh, PA 15261, USA

(Received 13 January 2005, revised 24 June

2005, accepted 25 July 2005)

doi:10.1111/j.1742-4658.2005.04878.x

Two novel rare mutations, MCAD
842GfiC (R256T) and MCAD
1166AfiG (K364R), have been investigated to assess how far the bio-chemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency When the gene for K364R was overexpressed in Escherichia coli, the syn-thesized mutant protein only exhibited activity when the gene for chapero-nin GroELS was co-overexpressed Levels of activity correlated with the amounts of native MCAD protein visible in western blots The R256T mutant, by contrast, displayed no activity either with or without chapero-nin, but in this case a strong MCAD protein band was seen in the western blots throughout The proteins were also purified, and the enzyme function and thermostability investigated The K364R protein showed only moder-ate kinetic impairment, whereas the R256T protein was again totally inac-tive Neither mutant showed marked depletion of FAD The pure K364R protein was considerably less thermostable than wild-type MCAD Western blots indicated that, although the R256T mutant protein is less thermo-stable than normal MCAD, it is much more thermo-stable than K364R Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein K364R, like several previously described MCAD mutant proteins, appears to be defective in folding R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity How the mutations specifically affect the catalytic activity and the folding is further discussed

Abbreviations

ACAD, acyl-CoA dehydrogenase; BCIP, 5-bromo-4-chloro indol-3-yl phosphate; DCPIP, 2,6-dichlorophenolindophenol; ETF,

electron-transferring flavoprotein; GCD, glutaryl-CoA dehydrogenase; INT, 2-(4-iodophenyl) 3-(4-nitrophenyl) 5-phenyl-tetrazolium chloride; IVD, isovaleryl-CoA dehydrogenase; NBT, 2,2¢-di-p-nitrophenyl 5,5¢-diphenyl 3,3¢-(3,3¢-dimethoxy-4,4¢-diphenylene) ditetrazolium chloride; PES, phenazine ethosulphate; SCAD, short-chain acyl-CoA dehydrogenase; VLCAD, very long chain acyl-CoA dehydrogenase.

lents from the fatty acyl substrate, and donating them

to electron-transferring flavoprotein (ETF), which

ulti-mately feeds them to the electron transport chain to

generate ATP [1] Within the active site, the substrate

fatty acyl chain is sandwiched between the

isoalloxa-zine ring of the FAD prosthetic group and the

carb-oxyl group of the catalytic glutamic acid residue

(E376) This residue removes one hydrogen as a proton

from the C-2 position of the fatty acid thioester The

other is simultaneously removed as a hydride ion by

the N-5 position of the isoalloxazine ring With this

oxidation step the FAD becomes reduced to FADH2

[2] The reducing equivalents are then transferred from

the reduced flavin of MCAD to ETF, and the

enoyl-CoA is released to be further degraded via the

b-oxida-tion cycle [1]

Defects in MCAD accordingly impair the ability to

degrade fatty acids, and MCAD deficiency is the most

frequently diagnosed clinical defect of fatty acid

meta-bolism [4], with a frequency of 1 : 15 000 in the USA

population [5] Eighty per cent of patients are

homo-zygous for the common K304E (MCAD
985AfiG)

mutation, and a further 18% have this mutation in

one of the two defective alleles [5] Symptoms of

MCAD deficiency cover a broad spectrum, ranging

from hypoglycaemia to seizures, coma and sudden

death, and usually present at a time of metabolic

decompensation associated with fasting or viral illness

[4] However, some individuals carrying the genetic

defect remain asymptomatic throughout life [6–8]

A variety of rare MCAD mutations have been

identified through screening programmes, and, where

they have been characterized at the protein level, the

defect has thus far mainly been in folding [9,10]

It is not yet clear whether this merely reflects a

greater statistical likelihood of impaired folding than

of impaired catalysis or cofactor binding Here we

describe two novel rare mutations with

contrast-ing enzymological consequences The first, R256T

(MCAD
842GfiC), was found as a compound

heterozygote with K304E in a screening programme

in the USA This newborn exhibited elevated

hexa-noyl, octanoyl and decenoyl carnitine levels,

indica-ting MCAD deficiency This mutation has previously

been reported in four siblings, who, though exhibiting

a biochemical profile indicative of MCAD deficiency

(i.e elevated hexanoylglycine in urine), have so far

remained clinically and developmentally normal [11]

The second mutation, K364R (MCAD
1165AfiG),

missense mutations affect the ability of the MCAD protein to fold, expression levels in Escherichia coli were monitored, with and without the co-overexpres-sion of GroEL and GroES [6,9,12] Stability of the mutant proteins in this model system was further investigated by determining the effect of temperature

on the enzyme activity and structure The MCAD mutants were also purified so that the kinetic parame-ters and stability of the homogeneous proteins could

be investigated Interaction with the natural electron acceptor, electron-transferring flavoprotein (ETF) was also tested, to give a more complete picture of the bio-chemical outcome of these point mutations

Results

The effect of chaperonin on the ability of the mutant proteins to fold

For a number of other MCAD point mutations, co-overexpression of the GroELS genes has been shown to rescue enzyme activity, suggesting that these mutations may affect folding in vivo [6,12] Therefore the GroELS genes were overexpressed with those for each of the MCAD mutants, K364R and R256T, and for the wild-type enzyme, in E coli cells grown both at

31
C and 37
C, and the effect on activity (ferri-cenium assay), and tetramer assembly (as determined

by western blot analysis of native gels) was investi-gated (Fig 1)

At 31
C, wild-type MCAD gave the highest level

of activity of the three proteins, with 9550 nmol ferriceniumÆmg)1Æh)1 (Fig 1A) This increased to

11 700 nmol ferriceniumÆmg)1Æh)1 in the presence of chaperonin, the moderate extent of this change pre-sumably reflecting successful unassisted folding for most of the MCAD In the absence of chaperonin, increasing the growth temperature to 37
C decreased the activity of wild-type MCAD by 55%, to 5340 nmol ferriceniumÆmg)1Æh)1 Co-expression of the chaperonin genes increased the MCAD activity nearly fourfold

to 19 100 nmol ferriceniumÆmg)1Æh)1, an increase of almost 60% when compared with growth at 31
C This figure may in part reflect upregulation of both MCAD and chaperonin It is clear, however, that, under these more stressful (though physiological) con-ditions of temperature, even the wild-type enzyme depends upon chaperonin for optimal folding in this recombinant overexpression system

No MCAD activity was detected in lysates of cells

expressing R256T, but, at both temperatures, the

fold-ing of this mutant protein appears to be even more

successful than for wild-type enzyme, as judged both

by the levels of MCAD tetramer present on the

west-ern blot and by the minimal effect of chaperonin

(Fig 1B) This suggests that the R256T mutation

harms the function of the folded protein rather than

the ability of the protein to fold

K364R, by contrast, appears to be a severe

tempera-ture-sensitive folding mutation In the absence of

chap-eronin, no activity was detected at either growth

temperature (Fig 1A) With chaperonin, the activity

expressed at 31
C (2950 nmol ferriceniumÆmg)1Æh)1)

could be rescued to 25% of the wild-type figure, but

on increasing the growth temperature this level

dropped to 6% (751 nmol ferriceniumÆmg)1Æh)1)

(Fig 1A) These diminished activities, as compared

with wild-type MCAD, clearly also correlate with

decreased amounts of MCAD protein detected in the

western blots (Fig 1B)

Comparison of activity levels of the mutant

proteins, as determined by activity staining

The apparent inactivity of R256T was further

investi-gated to determine whether this was specific to the

fer-ricenium assay, or whether this mutation renders the

enzyme entirely catalytically inactive Extracts of cells cultured at 31
C were run on native PAGE The sen-sitive activity stain, utilising phenazine ethosulphate (PES) and p-iodonitrotetrazolium violet (INT) as elec-tron acceptors, was used, and the gel was western blot-ted for direct comparison of the amount of folded tetramer with the activity level (Fig 2) In general, the activity staining correlated well with the corresponding ferricenium assay results (Fig 1A) and also compared well with the amount of folded tetramer present, for both wild type and K364R (Fig 2A) As the stain is

so sensitive, a slight staining could be seen for K364R

in the absence of chaperonin, even though the ferri-cenium assay detected no activity However, even with the increased sensitivity, R256T gave no indication of activity

Kinetic parameters of the purified variant proteins The mutant and wild-type proteins were purified to homogeneity by anion exchange and dye affinity chro-matography, with a novel substrate elution procedure proving very effective in securing a pure protein prod-uct The results of kinetic analysis are displayed as Michaelis–Menten plots in Fig 3A The Km, Vmax (as determined by the Direct Linear and Wilkinson meth-ods), and kcat values are shown in Table 1 The Km value of wild-type MCAD for octanoyl-CoA, 3.69 lm, compares well with the published value of 3.4 lm [13,14] Although the Km is increased somewhat for the K364R mutant protein (5.53 lm), the kcat is not significantly decreased R256T was also purified, but, even at a final concentration of 21.4 lgÆmL)1 in the assay, this mutant protein still showed no catalytic activity

The activity levels were also determined using the natural electron acceptor, electron-transferring flavo-protein (ETF) (Fig 3B), and, as expected, were very

A

B

Fig 2 Comparison of (A) western blot (showing the amount of tetramer formed) and (B) activity stain (showing the activity level of tetramer), of wild type (WT), R256T, and K364R MCAD both in the presence (+) and absence (–) of co-overexpressed chaperonin GroE

in E coli cells, grown at 31
C.

A

B

Fig 1 Comparison of wild type (WT), R256T, and K364R MCAD

both in the presence (+) and absence (–) of co-overexpressed

chap-eronin (GroE), in E coli, grown at 37 and 31
C (A) This shows the

activity levels as determined by ferricenium activity assay (error

bars ¼ standard deviation of the mean result) (B) Western blot

analysis of native PAGE gels, indicative of relative amounts of

sol-uble tetramer.

9% lower than wild type, suggesting that the primary effect of this mutation is not on the catalytic activity

of the enzyme

Absorption spectra of the purified mutant proteins

To assess whether these mutations affected the affinity

of the enzyme for the bound prosthetic group, FAD, absorption spectra were obtained As FAD absorbs strongly at 450 nm, the ratio A450⁄A280 gives a rough indication of the amount of FAD bound to the puri-fied protein (assuming no major change in the extinc-tion coefficient of the bound cofactor in the case of the mutants) [15] The peak absorbance ratios showed first of all that the wild-type enzyme as purified here (ratio¼ 7.2) is somewhat depleted of FAD because the value should be 5.2–5.5 This probably reflects the rigorous procedure to remove excess FAD added dur-ing the purification, and certainly does not indicate the level of contamination by other protein, to judge from SDS⁄ PAGE The ratios for R256T (8.8) and K364R (9.6), both handled in exactly the same way as the wild-type enzyme, suggest that both may be slightly weakened in their affinity for FAD, with K364R the most affected As this is an unstable protein, the FAD binding may be less secure than with a more tightly folded protein The catalytic competence of the enzyme

is in any case not greatly affected, suggesting that any FAD binding impairment cannot be the primary dele-terious effect of this mutation As the reduction in affinity is not as great for R256T, FAD depletion is clearly not responsible for the inactivity of the R256T mutant protein

Thermostability of the purified mutant proteins The effect of temperature on MCAD stability was directly investigated by incubating aliquots of each purified protein (10 lgÆmL)1) at various temperatures (4–55
C) for 10 min Each aliquot was also subject to western blot analysis, so that the activity could be compared with the amount of native tetramer present The thermostability curve (Fig 4A) shows that K364R

is less stable than wild-type, with 50% residual activity after 10 min at approximately 48
C, compared with

58
C for wild-type At 55
C K364R was completely inactive after 10 min R256T remained inactive at all temperatures The western blots compared well with

B

Fig 3 (A) Michaelis–Menten plot showing the substrate kinetics

of the purified mutant protein K364R and wild-type MCAD (WT).

(B) Comparison of the activity levels of purified K364R and

wild-type MCAD, as determined by both the ferricenium and ETF assays

(error bars indicate standard deviation of the mean result).

Table 1 K m (l M ), V max (nmol ferriceniumÆmg)1Æmin)1) (as

deter-mined by Direct Linear and Wilkinson methods), and kcat(s)1) for

octanoyl-CoA oxidation.

Km Vmax kcat Wild-type MCAD

Direct linear 3.69 24.4 · 10 3 19

Confidence limits (68%) 3.23–4.03 23.8–24.9 · 10 3

Wilkinson 3.68 24.6 · 10 3

19.1 Standard error 0.305 621

K364R

Direct linear 5.38 25.5 · 10 3

19.8 Confidence limits (68%) 5.09–5.87 25.4–25.6 · 10 3

Wilkinson 5.67 25.6 · 10 3 19.9

Standard error 0.186 274

the enzyme activity results (Fig 4B) Wild-type

MCAD shows a large amount of tetramer still present

at 55
C, at which temperature 68% enzyme activity

remains For K364R there was a dramatic decrease in

the tetramer level at 50
C, corresponding to the loss

of enzyme activity The R256T mutant protein,

although not as thermostable as wild-type, shows

con-siderably more structural stability than the K364R

mutant, with a higher amount of tetramer remaining

at 50
C

Sequence alignment and position of the

mutations in the three-dimensional structure

The acyl-CoA dehydrogenase (ACAD) family shares

30–35% sequence homology within a species, and each

individual ACAD member shows 85–90% sequence

identity between mammalian species [16] The

align-ment (not shown) of the human MCAD sequence with

published MCAD and short-chain acyl-CoA

dehydro-genase (SCAD) sequences from various other sources

shows that R256 is completely conserved across all the

species studied, indicating the importance of this

resi-due The three-dimensional structure of porcine MCAD, which differs in sequence from the human enzyme at fewer than 10% of the positions [17,18], has been solved to 2.4 A˚ (Fig 5) [17] In this structure, R256 is in very close proximity to the catalytic residue E376 During catalysis the E376 sidechain swings towards the Ca atom of the substrate, in order to abstract the proton [17] It seems likely that the fixed charge of the guanidino group of R256 stabilizes the catalytic carboxylate in the correct position for cata-lysis It is thus not surprising that removal of the con-served positive charge in the mutant R256T prevents catalysis Indeed this residue has recently been studied

in rat MCAD, where the arginine was mutated to alanine, lysine, glutamine and glutamic acid The authors found that the lysine mutant exhibited signifi-cantly reduced activity, whereas the other variants were completely inactive [19]

As expected from the extensive sequence homology, the recently solved structure of a mammalian SCAD shows a high degree of similarity to MCAD [20] At the amino acid position in SCAD corresponding to K364 in MCAD there is an arginine residue, R356 in the SCAD sequence It is striking that this conserva-tive substitution is identical to the clinical mutation in the present case Interestingly, though, this position in SCAD has also previously been identified as the site of

a clinical mutation R356W [20] This was found in

a female newborn, who presented with hypotonia, seizures and developmental delay [21] Another highly homologous ACAD, glutaryl-CoA dehydrogenase

Fig 4 Thermostability of purified wild-type (WT) MCAD and K364R

mutant protein (10 lgÆmL)1) over increasing temperature (A) This

shows the thermostability as determined by the ferricenium assay

after a 10-min incubation at the temperature indicated (error

bars ¼ standard deviation of the mean), and (B) the amount of

tetramer present, as determined by western blot analysis of native

PAGE gels.

Fig 5 Structure of pig MCAD, solved with bound octanoyl CoA [17], is modelled using NCBIs Cn3D programme (PDB: 3MDE) [38] The catalytic residue at position 376 and R256 are highlighted in yellow Also shown is the octanoyl CoA (8-CoA) and FAD.

valeryl-CoA dehydrogenase (IVD) (R363C), showing

both reduced stability and activity The activity was

undetectable with the PMS⁄ DCPIP assay, and only

0.01 lmol ETFÆmin)1Æ(mgÆprotein))1 when measured

using the highly sensitive ETF fluorescence quenching

assay [16,23] In very long chain acyl-CoA

dehydro-genase (VLCAD), an analogous mutation (R410H) has

also been found to cause disease in three compound

heterozygote patients [24,25] To the authors’

know-ledge, this residue is the most frequently mutated

posi-tion across the entire ACAD family Modeling of this

residue, using the rat SCAD crystal structure,

sugges-ted disruption of the local bonding and steric

hin-drance by the introduced amino acid

Discussion

In this paper we have described the protein folding,

enzyme function, and thermostability of two novel rare

MCAD mutants, R256T and K364R, which are

strik-ingly different in their molecular behavior The first

mutation, R256T is a nonconservative substitution of a

strongly basic internal residue by a smaller and less

polar one R256T was unaffected in its folding, with

lev-els of tetramer formation in E coli cells comparable to

wild type, even at 37
C Likewise there was only a

mod-erate decrease in the thermostability of this protein

Nevertheless, R256T is clearly an acute mutation,

because, regardless of its stability, the catalytic activity

was completely abolished, as determined by the

ferrice-nium assay, ETF assay, and the INT⁄ PES activity stain

As R256T was successfully purified by the same method

as wild-type MCAD, i.e using affinity elution with the

substrate, this mutation is unlikely to impede acyl-CoA

substrate binding, but rather must affect catalysis, either

in the acceptance of reducing equivalents from the

substrate, or the donation of these equivalents to ETF

Although K364R is a relatively conservative

substi-tution, exchanging one basic residue for another,

arginine is a bulkier residue, and may cause steric

hin-drance of the local structure of helix J, or affect the

interaction with neighboring helices of the C-terminal

domain K364R was found to be an acute,

tempera-ture-sensitive folding mutation from the chaperonin

studies The mutant protein was, at most, moderately

affected in its substrate kinetics, ETF interaction, and

FAD affinity when compared with wild type This

suggests that, although K364R is acutely affected in

its ability to fold into tetramer, whatever does fold

instability is not sufficient to impact greatly on the catalytic activity, as the kcat and the ETF interaction are relatively unaffected This mutation seems to affect mainly the initial folding and stability of the tetramer, and similarly lowered levels of tetramer in human cells would account for the observed elevation of indicator acyl-carnitine levels

From the biochemical studies, it would appear that both R256T and K364R, although showing very differ-ent effects at the protein level, are both severe muta-tions in terms of their overall effect on expressed activity As R256T has so far been found as a com-pound heterozygote with the K304E mutation, this could either mask or enhance the clinical manifestation

of the disease [6,26] Although the individuals with this mutation have remained asymptomatic, the analogous mutations in glutaryl-CoA dehydrogenase (R257W, compound heterozygote with P278S, and R257Q), are known to be disease-causing, indicating that R256T could also be potentially disease-causing [27] As K364R was found as a homozygote, our biochemical studies are more directly applicable

Regardless of the enzyme activity as determined by biochemical testing, the actual outcome can vary from individual to individual depending on the functional overlap of VLCAD, LCAD, MCAD and SCAD, the efficiency of the chaperonin-aided folding, the effi-ciency of the detoxification of accumulated intermedi-ates, and avoidance of exposure to the environmental triggers Certainly in the case of MCAD deficiency, environmental factors appear to outweigh the genetic factors [26] The possibility that different mutations alone may cause varying severity of disease, resulting

in the wide clinical manifestation, has been considered [28] However, subsequent biochemical and molecular folding studies of the various point mutations have revealed no clear correlation between the genotype and phenotype [6] This becomes most apparent in the study of the homozygous K304E, where the entire clin-ical spectrum of MCAD deficiency has been observed [5,6,26,29], suggesting that other background factors must modulate the severity of clinical presentation Therefore there is no correlation evident between the effect of the mutations, as determined experimentally for the protein, and the severity of disease precipita-tion Present evidence would suggest that, whilst the affected individuals in whom these new mutations were found have not shown overt clinical symptoms, these are nevertheless potentially dangerous mutations

Experimental procedures

Chemicals

5-Bromo-4-chloro indol-3-yl phosphate⁄ nitro blue

tetrazo-lium (BCIP⁄ NBT) tablets, octanoyl-CoA,

2,6-dichloro-phenolindophenol (DCPIP), INT, PES, ferrocene, sodium

hexafluorophosphate were all obtained from

Sigma-Ald-rich Ltd (Dorset, UK) Procion Red HE-3B textile dye

was a generous gift from Dr C.V Stead of the Dyestuffs

Division of Imperial Chemical Industries (Blackley,

Manchester, UK) Column chromatography media were

supplied by Amersham Biosciences UK Ltd (Amersham,

Buckinghamshire, UK) Filters for the Amicon Centricon

device were purchased from Millipore Ireland BV (Cork,

Ireland)

Mutagenesis and subcloning of R256T and K364R

The genes for the mutant MCAD proteins were

overex-pressed in E coli JM109 cells by using the pWT vector

(encoding the mature part of the human MCAD gene,

pre-ceded by an artificial methionine, under the control of the

lac operon) in which the MCAD
842GfiC (R256T) and

MCAD
1165AfiG (K364R) mutations were introduced by

site-directed PCR-based mutagenesis [12,30] Mutagenic

antisense oligonucleotides for MCAD
842GfiC (5¢-GAT

AAAACCACACCTGTAGTAGCTG-3¢) and MCAD

1165AfiG (5¢-CCTGTAGAAAGACTAATGAGGGATG

CC-3¢) (mutagenic substitutions are shown in bold), and

the antisense primer (5¢-GTAACGCCAGGGTTTTCCCA

GTCAC-3¢) were used to generate a megaprimer This was

used in a secondary PCR, with the sense primer (5¢-GATC

CAGATCCTAAAGCTCCTGCT-3¢), to generate the

full-length fragment, which was then subcloned into the pWT

vector, using the EcoRI and HindIII sites The expression

vectors were sequenced across the region encoding the

MCAD gene, to exclude PCR-based errors Each mutant

MCAD vector was cotransformed into JM109 with either

pGroESL (encoding the chaperones GroES and GroEL)

[31] or pCap (empty vector control), and cultured as

des-cribed elsewhere [12]

Polyacrylamide gel electrophoresis and western

blotting

SDS⁄ PAGE, native PAGE, and western blotting were

performed essentially as described previously, using

BCIP⁄ NBT tablets for colour development [9]

Protein purification

The mutant protein and wild type were purified by anion

exchange, and dye-affinity chromatography, as described

elsewhere [32]

Activity staining This method was initially modified from an activity assay for short chain acyl-CoA dehydrogenase [33] by substitu-ting a tetrazolium dye acceptor for DCPIP In the opti-mized protocol, native gels were submerged in the staining solution (50 mm glycine⁄ NaOH, pH 9.6, 1 mm p-iodo-nitrotetrazolium violet, 10 mgÆmL)1phenazine ethosulphate,

40 mm octanoyl-CoA) and placed on a shaker for approxi-mately 30 min until a strong colour developed Stain development was arrested by rinsing the gel with H2O

Enzyme kinetics Reaction rates were measured with concentrations of octa-noyl-CoA from 1 lm to 100 lm The reactions were carried out in 100 mm KPi, 5 mm EDTA buffer, pH 7.6 at 25
C with ferricenium as the final electron acceptor, as described elsewhere [34] The results were analysed using Enzpack 3 software (Biosoft) to determine the Kmand Vmaxvalues by the Direct Linear [35] and Wilkinson [36] methods The kcat

was then determined, using the MCAD monomer Mrvalues (46 590 for wild type, and 46 630 for K364R, the slight variation due to the mutation) to define the concentration

of active sites Michaelis–Menten plots were used only to display the results

Electron transferring flavoprotein (ETF) assay This assay utilizes 2,6-dichlorophenolindophenol as the final electron acceptor, in 50 mm KPi, 0.3 mm EDTA, 5% glycerol, pH 7.6 buffer, at 25
C [37]

Thermal stability of enzyme activity in cleared bacterial lysates

One hundred microlitre samples of bacterial lysates contain-ing mutant or wild-type MCAD (10 lgÆmL)1 in 100 mm

KPi, 5 mm EDTA, pH 7.6) were dispensed into separate Eppendorf flasks Each was incubated for 10 min in a water bath at the chosen temperature before removing to ice, and sampling for activity [34] and western blot analysis [12]

Acknowledgements

We warmly acknowledge the help and collaboration of

Dr Simon Olpin of the Sheffield Children’s Hospital who detected the patient with the MCAD
1166AfiG mutation and supplied material to make identification

of this mutation possible This work was supported by

a grant from the March of Dimes Foundation (grant number 1-FY-2003–688 to BSA) and also by Grant 1C⁄ 2002 ⁄ 073 under the International Collaboration Programme of Enterprise Ireland

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