(Received for publication, October 16, 1996, and in revised form, December 17, 1996)
From the This study was undertaken to identify the
cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize
all-trans-retinol and steroid alcohols in fetal tissues.
Degenerate oligonucleotide primers were used to amplify by polymerase
chain reaction 500-base pair fragments of alcohol dehydrogenase
cDNAs from chick embryo limb buds and heart. cDNA fragments
that encode an unknown putative alcohol dehydrogenase as well as the
class III alcohol dehydrogenase were identified. The new cDNA
hybridized with two messages of ~2 and 3 kilobase pairs in the adult
chicken liver but not in the adult heart, muscle, testis, or brain. The
corresponding complete cDNA clones with a total length of 1390 base
pairs were isolated from a chicken liver Cytosolic zinc-containing alcohol dehydrogenases
(ADH)1 with 40-kDa subunits are capable of
oxidizing a variety of primary, secondary, and aliphatic alcohols and a
limited number of cyclic alcohols (1). Six classes of dimeric ADH
isozymes have been identified in mammals (1). Except for class I, all
other classes of ADH have high Km values for ethanol
and oxidize medium-chain and long-chain alcohols most effectively.
Potential physiological substrates for ADH isozymes include retinoid
and steroid alcohols. Human class IV is the most efficient retinol
dehydrogenase, followed by class II and the class I The physiological significance of the cytosolic ADHs for steroid and
retinoid metabolism is not clear. The retinoid and steroid hormones
play a major role in fetal development and are detected in the
embryonal tissues during the early developmental stages. Since ADH
isozymes appear at different stages of embryogenesis, it is important
to determine which isozymes are present in the embryo during the stages
when retinoid and steroid hormones are synthesized. The chick embryo is
used as a model to study the effects of various hormones on gene
expression during development. In this study, we analyzed the mRNA
isolated from the chick fetal heart and limb buds at stage 21 for the
presence of messages encoding cytosolic ADH.
Degenerate
oligonucleotide primers were synthesized based on the peptide sequences
E(D/E)(I/V)EVAP and FGLGGVG, which are conserved in all animal alcohol
dehydrogenases (4). The first region corresponds to amino acids 24-30
(sense primer) and the second region to amino acids 198-204 (antisense
primer) of the human class I The liver, kidney, lung, heart, brain, skeletal muscle,
and bladder tissues were dissected from a 7-week-old chicken and frozen immediately in liquid nitrogen. Total RNA was isolated from each tissue
with RNAzol according to the manufacturer's protocol. Twenty micrograms of each RNA preparation were loaded onto a
formaldehyde-agarose gel and separated by electrophoresis. After
transfer to the Nytran filter (Shleicher & Schuell), the separated
mRNAs were hybridized with the [ A chicken liver The coding region of the new
ADH cDNA was amplified by PCR with the following primers:
CTCA The kinetic constants for retinol were determined by monitoring the
production of all-trans-retinal at 400 nm ( All kinetic studies were performed in 0.1 M sodium
phosphate, pH 7.4, at 25 °C with 2.4 mM NAD+
or 0.2 mM NADH. The kinetic constants for alcohols other
than retinol were obtained by monitoring the production of NADH at 340 nm ( The rabbit antiserum was raised against recombinant ADH-F. A 1:5,000
dilution of this antiserum detected 10 ng of purified ADH-F. Frozen
chicken liver was homogenized in 10 mM Tris-HCl, pH 7.4, plus 5 mM benzamidine and 1 mM dithiothreitol.
The homogenate was centrifuged at 10,000 × g for 15 min, and the supernatant was concentrated twice. Glycerol was added to
50% concentration, and the liver extract was stored at The amino acid substitutions occurring in chick ADH-F were model-built
into the human Sequences of human class I The pool of ~500-bp PCR products obtained with ADH-specific
primers from the limb buds and heart mRNA of chick embryos at stage
21 was subcloned in M13 vector, and 48 individual clones were
sequenced. Two of the clones from heart mRNA were found to have a
novel sequence with a high resemblance to ADH sequences, and 6 of the
clones from limb bud mRNA encoded a fragment that exhibited 87%
protein sequence identity with human The deduced protein sequence of the two novel identical PCR clones had
a high resemblance to ADH sequences but was different from that of the
The relationships of this presumed new ADH (ADH-F) with other ADH
isozymes were analyzed by progressive alignment (10). Table
I shows percentage identity of the new chick enzyme
(ADH-F) with other ADH classes (11) as well as the range of percentage identity of the isozymes from different species within the same ADH
class. The identity of the new ADH was highest with class I isozymes
(69%). Class II and VI ADH were the least similar (about 60%
identity) (Table I).
Intraclass isozyme variability and percentage amino acid identity of
ADH-F with other ADH classes
Department of Biochemistry and Molecular
Biology and the
Department of Medicine,
gt11 cDNA library. The
open reading frame encoded a 375-amino acid polypeptide that exhibited
67 and 68% sequence identity with chicken class I and III alcohol
dehydrogenases, respectively, and had lower identity with mammalian
class II (55-58%) and IV (62%) isozymes. Expression of the new
cDNA in Escherichia coli yielded an active alcohol
dehydrogenase (ADH-F) with subunit molecular mass of ~40 kDa. The
specific activity of the recombinant enzyme, calculated from active
site titration of NADH binding, was 3.4 min
1 for ethanol
at pH 7.4 and 25 °C. ADH-F was stereospecific for the 3
,5
-
versus 3
,5
-hydroxysteroids. The
Km value for ethanol at pH 7.4 was 17 mM compared with 56 µM for
all-trans-retinol and 31 µM for
epiandrosterone. Antiserum against ADH-F recognized corresponding
protein in the chicken liver homogenate. We suggest that ADH-F
represents a new class of alcohol dehydrogenase, class VII, based on
its primary structure and catalytic properties.
ADH (2).
Class IV and II ADH are not active with steroid alcohols, whereas class
I isozymes oxidize both retinoid and steroid substrates with relatively
low catalytic efficiency. Class I isozymes2
exhibit stereospecificity toward alcohol substrates. For example, horse
SS and human
ADH oxidize 3
-hydroxysteroids but not
3
-hydroxysteroids (3). ADH isozymes vary in their tissue
distribution; class IV ADH is expressed in the epithelial tissues of
mammals and is present in the human stomach mucosa and esophagus (1),
whereas class II
is found in fetal and adult liver. Class I
isozymes
1
1,
2
2,
3
3,
1
1,
2
2,
, and their heterodimers, as well as class II
, are the
predominant forms responsible for ethanol oxidation in the human adult
liver. Class I isozymes are also expressed to a lesser extent in
certain adult and fetal tissues, such as kidney, skin, gastrointestinal
tract, and lung.
PCR Amplification of ADH Isozymes
1 ADH. Four
oligonucleotides were synthesized for the sense orientation: 1),
GA(G/A)GA(T/C)GTIGA(G/ A)GTIGCICC; 2),
GA(G/A)GA(T/C)AT(A/C/T)GA(G/A)GTIGCICC; 3),
GA(G/A)GA(G/A)GTIGA(G/A)GTIGCICC; and 4),
GA(G/A)GA(G/A)AT(A/C/T)GA(G/A)GTIGCICC. Two oligonucleotides were synthesized for the antisense orientation: 1),
TT(T/C)GGICTIGGIGGIGTIGG; and 2), TT(T/C)GGITT(A/G)GGIGGIGTIGG.
Inosines were incorporated in all positions that required a degeneracy
of 4. Limb buds and hearts were dissected from 30 chick stage-21
embryos (3 days old). Total RNA was isolated from the pooled limb buds
and from the pooled hearts (RNAzol, Cinna/Biotecx Laboratories, Inc.,
Houston, TX). The total RNA from each pool was reverse transcribed and used for PCR amplification of ADH isozymes. PCR was performed with
various combinations of 4 sense and 2 antisense primers for 30 cycles
with annealing at 50 °C (1 min), extension at 72 °C (2 min), and
denaturing at 94 °C (1 min). Several combinations of primers
produced a ~500-bp product. Each ~500-bp band was isolated, reamplified, and subcloned into M13mp19RF (Life Technologies, Inc.)
vector for sequencing (U. S. Biochemical Corp.).
-32P]dATP-labeled
~500-bp PCR product in 50% formamide, 5 × Denhardt's solution, 5 × saline/sodium/phosphate/EDTA, 0.1 mg/ml salmon
sperm DNA, and 0.1% SDS at 42 °C overnight. After hybridization,
the filter was washed several times in 2 × SSC, 0.1% SDS at room
temperature, and the final wash was performed in 0.1 × SSC, 0.1%
SDS at 65 °C for 30 min.
gt11 cDNA library (Clontech) was screened with
the radiolabeled ~500-bp PCR product. The hybridization and washing
conditions were the same as those described for the Northern blot
analysis. Positive plaques were purified through three more rounds of
screening. The purified
phage was cleaved with EcoRI restriction endonuclease, and the cDNA insert was isolated and subcloned into M13mp19RF digested with EcoRI. Sense and
antisense single-stranded M13 DNA were prepared, and each was sequenced at least three times.
ATGGCCACTTCTGGAAAAGTT for the sense strand and
TGG
TCAGAAGAGCATCACGGTGC for the antisense strand. The
sense and antisense primers contained recognition sequences for the
restriction endonucleases BamHI and EcoRI
(underlined in the nucleotide sequence above), respectively. The
amplified coding region of the new cDNA was subcloned into the
expression vector pGEX-2T (Pharmacia Biotech Inc.). The final construct
encoded a 375-amino acid polypeptide fused with glutathione
S-transferase (GST). The expression of the fusion protein in
the E. coli TG-1 cells was performed as described for human
stomach
-ADH (5). Cells were harvested by centrifugation and
suspended in phosphate-buffered saline with Tween 80 (138 mM NaCl, 2.7 mM KCl, 1.2 mM
KH2PO4, 8.1 mM
Na2HPO4, pH 7.5, and 0.05% (w/v) Tween 80 (PBST)) containing 0.1%
-mercaptoethanol, 10 µM
ZnSO4, and the protease inhibitors phenylmethylsulfonyl
fluoride (50 µg/ml) and benzamidine (5 mM). The cells
were homogenized using a French press, and the insoluble fraction was
separated by centrifugation. The fusion protein was purified by
glutathione-agarose affinity chromatography. The alcohol dehydrogenase
activity of the recombinant protein was determined in a standard assay
containing 4.7 mM cinnamyl alcohol, 2.5 mM NAD+ in 0.1 M sodium phosphate, pH 7.4, at
25 °C. The GST domain was separated from chick ADH by cleavage with
human thrombin (Sigma). The efficiency of the cleavage was monitored by
the appearance of separate 40- (ADH) and 26-kDa (GST) protein bands in
SDS-polyacrylamide gel electrophoresis. ADH was purified from GST by
chromatography over S Sepharose and eluted with a NaCl gradient in 10 mM sodium phosphate buffer, pH 6.5, 10% glycerol, 2 mM dithiothreitol. Chick ADH eluted at 100 mM
NaCl. GST did not bind to the resin under these conditions. Glycerol
and dithiothreitol were found to stabilize enzyme activity. Therefore,
purified ADH was stored in 10 mM sodium phosphate, pH 7.4, 50% glycerol, and 2 mM dithiothreitol at
20 °C. The
concentration of glycerol was reduced to 10% before each experiment. Glycerol never exceeded 0.5% in the assay mixture, and this
concentration did not alter ADH-F activity measurements. The
concentration of ADH active sites was determined by observing
fluorescence (excitation wavelength at 328 nm and emission at 425 nm)
while titrating enzyme (1-2 mg/ml) with NADH in the presence of 99 mM isobutyramide in 10 mM sodium phosphate at
pH 7.4. The concentration of NADH binding sites was evaluated from the
intersection point of the linear regression of the fluorescence
titration above and below NADH saturation (6). The specific activity of
the chick ADH-F was calculated based on the concentration of NADH
binding sites. Total protein concentration was determined by a
dye-binding assay (Bio-Rad) using bovine serum albumin as a
standard.
= 29.5 mM
1 cm
1) (7). The retinol stock
solution was prepared in acetone, and aqueous retinol solutions were
prepared by dissolving the calculated amount of retinol stock solution
in 0.1 M sodium phosphate, pH 7.5, and 0.02% Tween 80. The
addition of 0.02% Tween 80 did not inhibit the enzyme activity. The
concentration of retinol in aqueous solution was determined by
measuring the absorbance at 328 nm (
= 39.5 mM
1 cm
1), and solutions were
used immediately.
= 6.22 mM
1 cm
1).
Reaction mixtures with steroid substrates contained 0.02% Tween 80. Steroid stock solutions were prepared in methanol, and concentration of
methanol in the assay mixtures was kept constant at 0.3 M. Chick ADH-F was neither active toward nor inhibited by methanol up to 3 M at pH 7.5. The Vmax and
Km values for alcohol substrates (at 2.4 mM NAD+) were calculated from a fit of the
kinetic data to the Michaelis-Menten equation, V = VmaxA/(Km + A), where A is the concentration of the varied
substrate. The kcat (min
1) was
obtained by dividing Vmax by the concentration
of active sites assuming a subunit Mr of 40,000. The apparent Km values for NAD+ and NADH
were determined with 1 mM cinnamyl alcohol and 100 µM cinnamyl aldehyde, respectively. The inhibition
constant for 4-methylpyrazole was determined with butanol as a
substrate by varying both butanol (68-200 µM) and
4-methylpyrazole (75-350 µM) concentrations. The Ki of 4-methylpyrazole was calculated from a fit of
the kinetic data to the equation for competitive inhibition,
V = VmaxB/(KB (1 + I/Kis) + B), where
B and I are butanol and 4-methylpyrazole concentrations, respectively (8). The Ki value for NADH was determined by varying NAD+ (15-60
µM) at 1 mM cinnamyl alcohol, using 0-10
µM NADH as the inhibitor.
20 °C. The
proteins in the chicken liver homogenate were separated by isoelectic
focusing using 3-10 pH gradient isoelectic focusing agarose plates
(FMC Bioproducts, Inc.). After focusing, the separated proteins were transferred to nitrocellulose membrane, blocked with 3% bovine serum
albumin in PBST, and incubated with a 1:5,000 dilution of antiserum.
The binding of anti-ADH-F antibodies was visualized with
125I-protein A.
1-structure using the molecular graphics program QUANTA (Molecular Simulations, Inc.). Following substitution of
all amino acid side chains in the dimer, the model structure was
subjected to 100 cycles of energy minimization using X-PLOR 3.1 with
the x-ray energy term omitted (9). The position for the epiandrosterone
molecule in the human
1-structure was found by manually
adjusting its position to minimize close contacts between the enzyme
active site and the substrate molecule.
,
, and
ADH; class I ADHs from the
alligator, cod, frog, horse E and S, mouse, ostrich, quail, rabbit, and
rat; class II ADHs from human and rat; class III ADHs from human,
horse, mouse, and rat; class IV ADHs from human and mouse; human
ADH6; and class VI from deer mouse were aligned with ADH-F
by a progressive alignment method according to Feng and Doolittle (10).
Sequences of ADHs were obtained from the GenBankTM.
-ADH (11, 12). The rest of the
clones contained cDNA sequences that were not related to ADH. Since
human class III ADH is not active with retinol, we did not pursue the
cloning and characterization of this
-ADH-like chick isozyme
further.
-ADH-like chick ADH and the chick class I ADH (13). A Northern blot
analysis of adult chicken tissues demonstrated that this partial PCR
product from embryonal heart hybridized with two messages of
approximately 2 and 3 kilobase pairs in adult chicken liver (Fig.
1). Other tissues (brain, testis, skeletal muscle, and
heart) did not show a detectable hybridization signal after 24 h
of exposure. A chicken liver
gt11 cDNA library was used to
isolate a full-length cDNA. Three independent clones hybridizing with the partial cDNA were isolated. Two clones encoded a complete cDNA, and one lacked the N terminus. The total composite cDNA sequence was 1408 bp long with the ATG starting codon at nucleotide 74 and the TGA stop codon at nucleotide 1202 (Fig. 2). The
open reading frame encoded a 375-amino acid mature polypeptide with predicted Mr of 40,016.
Fig. 1.
Northern blot analysis of adult chicken
tissues. 20 µg of total RNA from brain (lane 1),
testis (lane 2), muscle (lane 3), liver
(lane 4), and heart (lane 5) were separated by
size in the formaldehyde-agarose gel and transferred to Nytran
membrane. The membrane was hybridized with the 500-bp PCR product
encoding new chick ADH-F (panel A). The numbers on the
right refer to sizes of the mRNA ladder (kilobases).
Panel B shows the amount of ribosomal RNA present in each
lane.
[View Larger Version of this Image (72K GIF file)]
Fig. 2.
Nucleotide sequence and deduced protein
sequence of chick ADH-F. Numbers on the right
correspond to nucleotide sequence, and numbers on the left
correspond to amino acid sequence. The peptide regions that were used
to design degenerate oligonucleotides are underlined. The
starting Met is present at nucleotide 74 (MET). The amino
acid sequence is numbered from the Ala following the initiating Met
codon in accordance with numbering of other ADH isozymes. The
termination codon is indicated with an asterisk. The
residues discussed in the text are shown in reversed color (white on black background). The insertion of N
at amino acid 56 is shown in italic.
[View Larger Version of this Image (77K GIF file)]
ADH
class
Ia
IIb
IIIc
IVd
Ve
VIf
ADH-F
61 -69
54 -62
62 -64
62
-63
63
57
I
69 -83
53
-61
62 -66
66 -71
59 -67
55 -60
II
66
-72
55 -63
51 -58
54 -60
47 -51
III
93
-95
60 -61
58
55
IV
87 -89
60a
56 -57
V
100b
67a
VI
100c
a
Includes isozymes from human ( ,
1, and
1), horse (S and E), rat, mouse, rabbit, chicken, ostrich,
alligator, and frog.
b
Includes isozymes from human, rat, and ostrich.
c
Includes isozymes from human, horse, rat, and mouse.
d
Includes isozymes from human, rat, and mouse.
e
A single representative of the class, human ADH6,
is known.
f
Class VI is represented by the deer mouse isozyme.
To characterize the catalytic properties of the new isozyme, the ADH-F
cDNA was expressed in E. coli as fusion protein with GST
(14). The recombinant enzyme separated from GST by thrombin cleavage
had an apparent subunit Mr of 40,000 on
SDS-polyacrylamide gel electrophoresis. 1 to 2 units of activity were
obtained from a 1-liter culture, which corresponded to 12-24 mg of
active enzyme. The specific activity of the ADH after thrombin cleavage
was the same as that for the ADH-GST fusion protein. Specific activity was determined utilizing fluorescence active site titration by directly
measuring the concentration of NADH binding sites. The Km value of the new ADH for ethanol was relatively
high, 17 mM, and the kcat value was
3.4 min1 (Table II). The
Km values were several orders of magnitude lower for
long-chain and large hydrophobic alcohols than for ethanol (Table II).
The Km for cinnamyl alcohol was 8.4 µM, and the
kcat/Km value was 580 min
1 mM
1. The apparent
Km values for NAD+ and NADH were 5.4 and
5.3 µM, respectively (Table II). Inhibition of
NAD+ reduction by NADH with cinnamyl alcohol held constant
at 1 mM was consistent with competitive inhibition. The
Ki for NADH was 4.0 ± 0.5 µM.
Inhibition of butanol oxidation by 4-methylpyrazole also fitted best
the competitive inhibition model. The Ki of
4-methylpyrazole was 300 ± 50 µM. These data are
consistent with results for horse liver ADH (15, 16) and an Ordered Bi Bi mechanism.
|
The secondary alcohol (R)-()-2-butanol had a
kcat/Km value seven times
greater than (S)-(+)-2-butanol (Table III). The catalytic efficiencies with all-trans-retinol (Table
III) and 3
-hydroxy-5
-steroids (Table IV) were
about 102 times higher than with ethanol, due primarily to
the much lower Km values (Tables II and IV). The
catalytic efficiency with the steroid alcohols epiandrosterone,
dehydroepiandrosterone, and dihydrotestosterone was similar to that
with retinol (Tables III and IV). No activity was observed with steroid
alcohols having a hydrogen in the 5
configuration
(3
-hydroxy-5
-androstan-17-one or 5
-pregnenolone) (Table IV).
Stereospecificity of the new ADH for 3
-hydroxy-5
-steroids was
confirmed in the reverse reaction. ADH-F was active in reducing the
steroid aldehyde 5
-androstan-17
-ol-3-one with NADH, but it was
not active with 5
-androstan-17
-ol-3-one (Table IV).
|
|
The protein corresponding to the wild-type ADH-F was detected in the
chicken liver homogenate with the rabbit antiserum raised against
recombinant ADH-F (Fig. 3). This antiserum cross-reacted with 100 ng of human class I, II, and IV but not class III ADH proteins
(not shown), all of which have similar subunit molecular weights and
cannot be separated by SDS-polyacrylamide gel electrophoresis. Thus,
isoelectric focusing was employed to separate the 80-kDa dimers of
chick ADH isozymes according to their isoelectric points. Recombinant
ADH-F appeared as a smear of multiple protein bands with pI values
ranging from 7.1 to 8.0 (Fig. 3, lane 1). All bands exhibited activity with 100 mM ethanol, 5 mM
trans-2-hexen-1-ol, and 100 µM
3-hydroxysteroid alcohols. Protein bands binding anti-ADH-F antibodies also appeared in the chicken liver homogenate separated by
isoelectric focusing in the same range of pI values as the recombinant
ADH-F (Fig. 3, lanes 2 and 3).
This study was undertaken to identify the isoforms of cytosolic
40-kDa (subunit) ADH in chick fetal tissues that oxidize the retinoid
and steroid alcohols. The only ADH-related PCR product amplified from
limb bud mRNA was identified as chick class III ADH based on the
87% protein sequence identity with human class III -ADH (12).
Because human class III ADH is not active with retinol, we were not
interested in characterizing the corresponding chick enzyme. However,
PCR amplification of embryonal heart mRNA yielded a novel
ADH-related cDNA. This new cDNA encoded an active enzyme named
ADH-F.
Northern blot analysis of tissues from a 7-week-old chicken showed that
among seven tissues analyzed, only the liver contained an mRNA
hybridizing with the partial PCR product from the fetal heart. The
adult heart mRNA did not contain a message that hybridized with
this PCR product. A change in the tissue-specific expression of ADH
gene between fetal and adult organism was also observed for the class I
ADH mRNA in rat (17) and may reflect different metabolic needs of
the tissues at different stages of development. The complete cDNA
isolated from the chicken liver cDNA library encoded an ADH (ADH-F)
with the polypeptide size similar to those of other ADH isozymes (375 amino acids without the starting methionine) (Fig. 2). ADH-F contained
13 residues (Fig. 2) that are conserved in the 47 members of the
zinc-containing ADH family excluding -crystallin (4). Sequence
alignment with other animal ADHs indicated a single amino acid
insertion after position 55. Therefore, the numbers of the conserved
amino acid residues after Gly-55 were shifted by one position in chick
ADH-F (Fig. 2) when compared with class I ADH (18). The conserved
glycines and the valine of the substrate binding domain were present at
positions 67, 72, 78, 87, and 81 (Fig. 2). The four glycines of the
coenzyme binding domain were in positions 193, 202, 205, and 237. Conserved ligands to the catalytic zinc, Cys-46 and His-68, were also
present. The new ADH-F sequence had Asp-224, which has been suggested
to determine the specificity for NAD+ versus
NADP+ as a coenzyme, and Thr-48, which is thought to form a
hydrogen bond with the alcohol hydroxyl group bound to the catalytic
zinc (19). Cysteines 98, 101, 104, and 112, which are responsible for
binding the noncatalytic zinc (20), were conserved in the new ADH.
However, the sequence of this ADH had less than 68% identity with any
of the known ADH isozymes (Table I); hence, we conclude that ADH-F
belongs to a separate class in the family of ADHs, class VII.
The new ADH gene encoded an active enzyme when produced as a recombinant protein in E. coli. Antiserum against this recombinant ADH-F recognized protein bands in the chicken liver homogenate with the same range of isoelectric points as the multiple ADH-F forms (Fig. 3). The slightly more basic pI of the recombinant protein is consistent with the lack of N-acetylation in E. coli-expressed proteins (21).
The functional and kinetic properties of the new recombinant ADH-F were
compared with those of other ADHs. The yield of ADH-F protein was high
(up to 14 mg/L of E. coli culture), but the specific activity with saturating ethanol at pH 7.4 was relatively low (0.08 unit/mg), a value that is similar to that of the human class I
1-ADH (0.1 unit/mg) (22). The Km
value for ethanol (17 mM) was close to that of human
stomach class IV ADH (29 mM) (5) and human liver class II
ADH (34 mM) (23) (Table II). NADH inhibited
NAD+ reduction competitively, and 4-methylpyrazole was a
competitive inhibitor of butanol. These inhibition results are
consistent with the Ordered Bi Bi mechanism suggested for other ADHs.
ADH-F sensitivity to 4-methylpyrazole inhibition was similar to that of
the human class IV ADH (Ki = 350 µM)
(5). The Ki value (300 µM) was greater
than that of class I and less than that of class II ADH.
The catalytic efficiency of ADH-F toward the secondary alcohol
(R)-()-2-butanol was seven times that of
(S)-(+)-2-butanol. This specificity appeared to be similar
to that of the human
isozyme, where its catalytic efficiency was
about four times higher with (R)-(
)-2-butanol (136 min
1 mM
1) than with
(S)-(+)-2-butanol (37.6 min
1
mM
1) (24). It has been suggested that the
specificity of ADHs toward secondary alcohols is affected by amino
acids at positions 48 and 93. Chick ADH-F has Thr-48 as in
ADH
and a unique Pro at the position homologous to residue 93 (94 in
ADH-F). Modeling of the amino acid substitutions present in chick ADH
shows that Pro can easily be accommodated at position 93 (Fig.
4) and that the region of the active site occupied by
the secondary alcohol more closely resembles
, with Ala at
position 93, than
1
1, with Phe at
position 93. Thus, it is not surprising that this new ADH isozyme
possesses a stereospecificity for small secondary alcohols that is more
similar to
than
1
1 (or horse ADH). The catalytic efficiency
(kcat/Km) of ADH-F was higher for oxidation of large alcohols. For example, ADH-F was 5 × 103 times more efficient with trans-2-hexen-1-ol
than with ethanol.
Chick ADH-F was similar to human class I isozyme in that it
oxidized both retinoid and steroid alcohols (Tables III and IV). It was
different from the other two retinol-oxidizing ADH isozymes, class IV
and class II
, which were not active with steroids (5,
25). ADH-F oxidized epiandrosterone about 88 times more slowly than
horse SS ADH (0.51 min
1 versus 44 min
1) (26). The catalytic properties of ADH-F suggest
that it may function as a steroid/retinoid dehydrogenase in chick.
However, the physiological significance of chick ADH-F for steroid and retinoid metabolism will be clarified once the tissue-specific expression pattern during development and the amount of the active enzyme in tissues are determined.
In general, no single amino acid difference appears to be responsible
for the unique kinetic properties of the new chick ADH. The ability to
oxidize large hydrophobic alcohols, such as retinol and
3,5
-hydroxysteroids, appears to be the result of several amino
acid substitutions near the active site zinc atom and at the entrance
to the alcohol binding pocket. With current knowledge, the ability of
ADH isozymes to oxidize 3
-hydroxysteroids has been limited to those
that possess a Ser at position 48 (human
and horse SS ADH).
ADH-F has a Thr at position 48. Its ability to bind steroids
productively may be due to substitutions in the vicinity of position
93. In sterol-oxidizing horse SS and human
, which have a Ser at
position 48, there is also a Phe at position 93, which is usually
preceded by a Pro-Leu sequence. The sequence Leu-Phe-Pro in chick
ADH-F, instead of Pro-Leu-Phe as in most class I isozymes, may account
for the difference in steroid alcohol specificity. In addition, there
are unusual substitutions at positions 318 and 319, where Leu and Ala
substitute for Ile and Phe, respectively. Thus, the "floor" of the
alcohol binding pocket appears to be more open in ADH-F compared with
class I
1
1, and this could make the site
more accessible to large substrates (Fig. 4). The enzyme appears to be
sensitive to the configuration at the 5-position of steroid alcohol,
since the 5
-hydroxysteroid alcohols are inactive (Table IV).
Molecular modeling suggests that the stereospecificity at the
5
-position of the sterol may be due to the presence of the extra
methyl group of Thr-48 and the rearranged floor of the substrate
binding pocket in the ADH-F compared with
1
1 (Fig. 4).
The productive association of large hydrophobic substrates leading to
efficient oxidation is usually associated with rearrangements near the
entrance to the alcohol binding pocket. It was shown that both the
horse SS and the human class IV isozyme efficiently oxidize
sterols and retinol, respectively, due to alterations in the loop at
the entrance to the alcohol binding site comprising residues 112-119
(5, 27). Both of these isozymes possess single amino acid deletions
that appear to widen the mouth of the substrate binding site,
permitting easier access for these large substrates. Although chick
ADH-F isozyme does not possess such a deletion, the presence of His and
Trp in positions 115 and 142, respectively, may affect the conformation
of this loop. A neutral or acidic residue at position 115 helps to
correctly position this loop by hydrogen bonding with the peptide
nitrogen of residue 118 in most class I ADH crystal structures. The His at position 115 will not perform the same function to anchor this loop
structure in place, and a conformational change in the structure of
this loop could create a more open substrate binding site. The
substitution of Asp-115 by Trp in the cod ADH crystal structure appears
to be the primary reason for the conformation of this loop to adopt an
-helical structure (28). It is not clear whether the insertion of
one amino acid in the region between positions 55 and 60 will also
affect the structure at the entrance to the substrate binding site.
Mutagenesis in class III
-ADH, which also has an insertion in this
region, strongly implicates a role for Asp-57 in binding of the
substrate hydroxymethyl glutathione (29). Our modeling of chick ADH
would suggest that Phe-57 could form favorable van der Waals contacts
with the hydrophobic face of hydroxysteroids.
Another interesting substitution occurs at position 173. In most ADH isozymes the catalytic zinc ligand Cys-174 is surrounded by two glycines. These glycines may provide the necessary flexible linkage between the catalytic and coenzyme binding domains to allow the large conformational change observed upon coenzyme binding. The presence of Ala at this position may impair the ability of this isozyme to undergo rapid conformational shifts in its structure and may explain, at least in part, the relatively low turnover rate of this isozyme.
Thus, ADH-F appears to be unique in terms of its structure-function
relationships. This enzyme has low specific activity; it is active with
3,5
-hydroxysteroids but not with 3
,5
-hydroxysteroids; it is
active toward steroid substrates in the absence of Ser-48; and it is
active toward retinol in the absence of deletion in the loop between
amino acids 115 and 120. Several amino acid substitutions discussed
above suggest an explanation for some of its properties. X-ray
structure determination of the enzyme will provide more complete
insight into the structural basis of its substrate specificity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U73654[GenBank].