From the
Mutations in the
The energy-transducing nicotinamide nucleotide transhydrogenases
constitute a class of membrane-bound enzymes which catalyze the
reaction shown in Equation 1.
On-line formulae not verified for accuracy
The bovine enzyme is a homodimer of monomer M
The
transhydrogenases of Escherichia coli and Rhodobacter
capsulatus are each composed of two unlike subunits,
We have shown earlier that the bindings of NADPH and
NADP, but not of NADH and NAD, alter the conformation of the bovine
transhydrogenase in different ways and have argued on the basis of both
theoretical considerations and experimental results that
inside-to-outside proton translocation by the enzyme (reversal of
Equation 1) is accomplished through substrate-induced conformation
change of the protein and is paid for by the difference in the binding
energies of substrates (NADPH + NAD) versus products
(NADP + NADH). Because NADPH and NADP binding, but not NADH and
NAD binding, results in detectable conformation changes of the bovine
transhydrogenase, we further proposed that it is mainly the difference
in the binding energies of NADPH and NADP that drives inside-to-outside
proton translocation via protein conformation changes (Yamaguchi and
Hatefi, 1989; Yamaguchi et al., 1990; Hatefi and Yamaguchi,
1992).
The five transhydrogenases whose amino acid sequences are
known contain regions of high sequence identity in the NAD(H) and
especially the NADP(H) binding domains as well as in a 19-residue-long
and a 58-residue-long stretch in the hydrophobic domain.
9-Aminoacridine
fluorescence was measured at 37 °C by an SLM photon-counting
fluorescence spectrophotometer at the excitation wavelength of 420 nm
and the emission wavelength of 500 nm. To the reaction mixture (2.0
ml), containing 10 mM Tricine/NaOH (pH 8.0), 2.5 mM MgSO
Free carboxyl groups in the membrane-intercalating domains of
proton-translocating enzymes are generally considered as good
candidates for participation in transmembrane proton translocation,
witness Asp-85 and Asp-96 of bacteriorhodopsin (Rothschild, 1992) and
Asp-61 of subunit c of E. coli ATP synthase complex
or the corresponding Glu in ATP synthases from other species
(Fillingame, 1990). As mentioned earlier, there is in the
400-residue-long hydrophobic domain of the transhydrogenase subunits a
single conserved aspartic acid residue (E. coli
For this purpose, it was
considered important to express the mutated forms of the enzyme in a
strain of E. coli from which the wild-type transhydrogenase
genes had been deleted (TH
The cyclic transhydrogenase reaction was of
interest to us, because it appeared to test the functionality of the
catalytic sector of the enzyme without the constraints that an impaired
proton channel might exert on the normal NADPH
It has been shown here that the mutation in E. coli of the only conserved dicarboxylic acid residue in the hydrophobic
domain of the
It has also been shown here that, in addition
to 90% inhibition of NADPH
Another finding of interest, especially in the case of the
The
apparent K
The apparent S
We thank Drs. Takao Yagi and Akemi Matsuno-Yagi for
fruitful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit of Escherichia coli proton-translocating nicotinamide nucleotide transhydrogenase of
the conserved residue
Asp-213 to Asn (
D213N) and Ile
(
D213I) resulted in the loss, respectively, of about 70% and 90%
NADPH
3-acetylpyridine adenine dinucleotide (AcPyAD)
transhydrogenation and coupled proton translocation activities.
However, the cyclic NADP(H)-dependent NADH
AcPyAD
transhydrogenase activities of the mutants were only
35%
inhibited. The latter transhydrogenation, which is not coupled to
proton translocation, occurs apparently via NADP under conditions that
enzyme-NADP(H) complex is stabilized. Mutations
D213N and
D213I also resulted in decreases in apparent K
for the NADPH
AcPyAD and
S
(NADPH concentration needed for
half-maximal activity) for the cyclic NADH
AcPyAD
transhydrogenation reactions, and in K
,
as determined by equilibrium binding studies on the purified wild-type
and the
D213I mutant enzymes. These results point to a structural
role of
Asp-213 in energy transduction and are discussed in
relation to our previous suggestion that proton translocation coupled
to NADPH
NAD (or AcPyAD) transhydrogenation is driven mainly by
the difference in the binding energies of NADPH and NADP.
= 109,065 (Yamaguchi et al., 1988). The monomer
is composed of 3 domains, a hydrophilic 430-residue-long
NH
-terminal domain that binds NAD(H), a hydrophobic
400-residue-long central domain that is largely membrane-intercalated
and harbors the proton channel of the enzyme, and a hydrophilic
200-residue-long COOH-terminal domain that binds NADP(H) (Yamaguchi et al., 1988; Yamaguchi and Hatefi, 1991; Hatefi andYamaguchi,
1992). The enzyme exhibits half-of-the-sites reactivity. It binds 1 mol
of NADH and 1 mol of NADPH per dimer (Yamaguchi and Hatefi, 1993), and
its transhydrogenase activity is completely inhibited by covalent
modification with 1 mol of
[
C]N,N`-dicyclohexylcarbodiimide
(Phelps and Hatefi, 1984; Wakabayashi and Hatefi, 1987a) or 1 mol of
[
H]FSBA
(
)(Phelps and
Hatefi, 1985; Wakabayashi and Hatefi, 1987b) per dimer.
with M
53,000 and
with M
48,000 (Clarke et al., 1986; Lever et
al., 1991; Ahmad et al., 1992). The E. coli enzyme has been shown to be an
heterotetramer (Hou et al., 1990). The transhydrogenase
of Rhodospirillum rubrum chromatophores has 3 subunits,
designated
1,
2, and
for consistency with the earlier
subunit designations of the E. coli enzyme (Yamaguchi and
Hatefi, 1994; see also Williams et al.(1994)). The molecular
masses of the R. rubrum transhydrogenase subunits are
1
= 40.3 kDa,
2 = 14.9 kDa, and
= 47.8
kDa. Subunit
1 is water-soluble and easily detached from
chromatophores. Together the two subunits of the E. coli or
the three subunits of the R. rubrum enzyme (the Rb.
capsulatus transhydrogenase has not been sequenced) exhibit the
same tridomain hydropathy profile as the bovine enzyme (Yamaguchi and
Hatefi, 1994). In addition, the predicted amino acid sequences of
putative single-subunit transhydrogenases from Eimeria tenella (Kramer et al., 1993; Vermeulen et al., 1993)
and Entamoeba histolytica (Yu and Samuelson, 1994) exhibit a
similar hydropathy profile, except that they could be described as
-tail-to-
-head spliced versions of the E. coli enzyme.
(
)The 58-residue-long stretch is adjacent to the
hydrophilic domain that binds NADP(H) (Fig. 1). Because such
stretches of high sequence identity in the hydrophobic domains of
related enzymes are rare, we felt that these membrane-intercalating
regions may have functional significance with regard to the
enzyme's proton translocation activity. In the entire
400-residue-long hydrophobic domains of the five sequenced
transhydrogenases, there are only 4 conserved charged residues and 3
conserved histidine residues. In the 19-residue-long stretch of high
sequence identity, there is a conserved histidine residue (E. coli
His-91, see Fig. 1) which when mutated in the E.
coli enzyme to Ser, Thr, or Cys results in
90% loss of
transhydrogenation and proton pumping activities (Holmberg et
al., 1994). As seen below, there is in the 58-residue-long stretch
a conserved aspartic acid residue (E. coli
Asp-213, see Fig. 1) whose mutation in the E. coli enzyme to Asn
resulted in our hands in
70% activity loss (see also Holmberg et al.(1994)), but its mutation to Ile caused 90% loss of
transhydrogenase and coupled proton translocation activities. Neither
mutation greatly inhibited the NADP(H)-dependent cyclic NADH
AcPyAD transhydrogenation activity of the enzyme, which involves both
the NAD and the NADP binding sites, but is not coupled to proton
translocation (see below). However, the
D213I mutation increased
the affinity of the enzyme for NADPH by 3-fold. The implications of
these results with regard to energy transduction by the
transhydrogenase will be discussed.
Figure 1:
Locations in the subunit of E. coli transhydrogenase and compositions of the
19-residue-long and the 58-residue-long hydrophobic stretches of high
sequence identity. The top rectangles depict that
and
the
subunits of the E. coli transhydrogenase, with their
hydrophilic NAD(H) and NADP(H) binding domains (unshaded) and
their hydrophobic domains (shaded). The amino acid sequences
of the 19- and the 58-residue-long stretches of high sequence identity (heavily shaded areas) are shown below the rectangles for the
5 sequenced transhydrogenases. The locations of
His-91 (H91) and
Asp-213 (D213) are
marked.
Materials
NADPH, NAD, and ATP were obtained from
Calbiochem; AcPyAD and NAD kinase were from Sigma; asolectin was from
Associated Concentrates; Ultrogel AcA34 was from IBF Biotechnics;
DEAE-Bio-Gel A agarose and Muta-Gene Phagemid In Vitro Mutagenesis Kit were from Bio-Rad; Sequenase version 1.0,
nucleotide kit for DNA sequencing with 7-deaza-dGTP, and random-primed
DNA labeling kit were from U. S. Biochemical Corp., and
[4-H]NAD (1200 mCi/mmol) was from Amersham.
Bacterial strains and plasmids were obtained from the following
sources: E. coli strain MC4100 (F
, araD139,
(arg F-lac)U169,
ptsF25, relA1, flb5301, rpsL 150.
) from ATCC;
pBluescript II KS(+) from Stratagene; pUC4K plasmid from Pharmacia
Biotech; pLC10-19 from the Clarke and Carbon Collection, carrying
the E. coli transhydrogenase gene (Clarke and Bragg, 1985),
was supplied by Dr. B. Bachman (E. coli Genetic Stock Center,
Yale University); pDC21, a derivative of pUC13, carrying the E.
coli transhydrogenase gene (Clarke and Bragg, 1985), was the
generous gift of Dr. P. D. Bragg, University of British Columbia; and
pMAK705, whose replication is temperature-sensitive and carries the
chloramphenicol resistance gene (Hamilton et al., 1989), was
kindly supplied by Dr. S. R. Kushner, University of Georgia.
Construction of Transhydrogenase Deletion
Mutant
Basic recombinant DNA techniques were performed according
to Sambrook et al.,(1989). From pLC10-29, a 4.8-kilobase HindIII fragment carrying the transhydrogenase coding region
was excised and inserted into the HindIII site of pBluescript
II. By digestion of the constructed plasmid with HpaI, a
2.7-kilobase fragment, covering most of the transhydrogenase coding
region, was cut out and replaced by the kanamycin resistance gene
cartridge from pUC4K. Then the whole insert was excised with EcoRV and inserted into the HindIII site of pMAK705
(pMAKTK). As described by Hamilton et al.(1989), pMAKTK was
integrated into the chromosomal DNA of E. coli strain MC4100,
and then the transhydrogenase genes were replaced by the kanamycin
resistance gene (strain MC4100 TH). The gene
replacement was confirmed by Southern blotting (Southern, 1975).
Site-directed Mutagenesis
The transhydrogenase
genes were removed from pDC21 by double digestion with BamHI
and HindIII and ligated with pTZ18U digested with BamHI and HindIII. With this pTZ18U plasmid,
site-directed mutagenesis was carried out to convert Asp-213 to
Asn or Ile, using the reagents and protocol as outlined in the Bio-Rad
Muta-Gene Mutagenesis Kit (Kunkel et al., 1987). The plasmid
DNA was prepared from individual colonies, and the mutants were
identified by double-strand DNA sequencing (Chen and Seeburg, 1985).
After confirming the DNA sequence, each Tth111I-BssHII 450-base pair DNA fragment was excised
and replaced with the counterpart of pDC21.
Enzyme Assays
Transhydrogenation from NADPH to
AcPyAD was assayed spectrophotometrically at 375 nm at 37 °C in a
reaction mixture containing 50 mM sodium phosphate (pH 7.0),
0.01% Brij 35, 10 µg of lysophosphatidylcholine, and 0.5 mM each of NADPH and AcPyAD. An extinction coefficient of 6.1
mM cm
was used to
calculate the rates. Transhydrogenation from NADH to AcPyAD in the
presence of NADP or NADPH was measured spectrophotometrically at 375 nm
at 37 °C in a reaction mixture containing 10 mM MES/KOH
(pH 6.0), 10 µg of lysophosphatidylcholine, 0.2 mM NADP or
NADPH, and 0.2 mM AcPyAD. The reaction was started by the
addition of 20 µM NADH. Protein concentration was measured
by the method of Peterson(1977). One unit of activity is defined as 1
µmol of AcPyAD reduced by NADPH per min.
, 0.25 mM dithiothreitol, and 0.2 mM EDTA, were added 20 µg of liposome-reconstituted
transhydrogenase and 1 µM 9-aminoacridine followed by 0.2
mM NADPH. Then, 0.4 mM AcPyAD and 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone were
added as indicated, and the fluorescence changes were monitored at 500
nm.
Culture of E. coli Cells and Preparation of
Membranes
E. coli strain MC4100 TH was transformed with pDC21 or its mutated plasmids. Each single
colony was inoculated into the LB medium containing ampicillin (100
µg/ml). Cells were grown until the late logarithmic phase,
collected by centrifugation at 8,000 rpm for 10 min (Sorvall GSA
rotor), and washed with 0.9% NaCl. The cells (wet weight, 14 g) were
then suspended in 140 ml of 50 mM Tris-HCl (pH 7.8),
containing 1 mM EDTA and 1 mM dithiothreitol (TED),
and sonicated in a Branson sonifier at output 8 and 25% pulse for 10
min. Unbroken cells were removed by centrifugation at 8,000 rpm for 10
min, and membranes were collected by centrifugation at 39,000 rpm for
45 min (Beckman 42Ti rotor). Membranes were suspended in a small volume
of TED and homogenized.
Purification of Enzymes
E. coli membranes
(30 mg of protein) were suspended in 6 ml of TED, and 1.5 ml of 10%
Triton X-100 was slowly added. After stirring for 15 min at room
temperature, the sample was centrifuged at 48,000 rpm (Beckman 50Ti
rotor) for 45 min. The supernatant (15 ml) was directly loaded on a
DEAE Bio-Gel A agarose column (1.5 14 cm) equilibrated with
TED, containing 0.125 M NaCl and 0.02% potassium cholate. The
enzyme was eluted by a linear NaCl concentration gradient from 0.125 M to 0.5 M in TED, containing 0.02% potassium
cholate. Approximately 1-ml fractions were collected. The active
fractions eluted at 0.20 M to 0.23 M NaCl were
combined and concentrated to 1.0 ml by a Centricon-30 concentrator and
loaded on an Ultrogel AcA34 column (1.5
68 cm) equilibrated
with TED, containing 0.02% potassium cholate. Active fractions were
combined (10 ml) and concentrated to
700 µl by a Centricon-30
concentrator.
Reconstitution of the Purified Enzymes into
Liposomes
Essentially the protocol developed by Sone et
al.(1977) was followed. Asolectin (200 mg) and cholic acid (150
mg) were added to 10 ml of 10 mM Tricine/NaOH (pH 8.0),
containing 5 mM dithiothreitol and 0.2 mM EDTA. After
adjustment of pH to 8.0 with 1 M KOH, the mixture was
sonicated to clarity. The purified enzyme (100 µg of protein) was
added to 2 ml of the asolectin suspension, and the mixture was dialyzed
against 1 liter of 10 mM Tricine/NaOH (pH 8.0), containing 2.5
mM MgSO, 0.25 mM dithiothreitol, and 0.2
mM EDTA for 20 h.
Preparation of
[4-
[4-H]NADPH
H]NADP
was prepared by phosphorylation of [4-
H]NAD (1200
mCi/mmol) in the presence of ATP and NAD kinase essentially according
to Wang et al.(1954). [4-
H]NAD and
[4-
H]NADP were separated on a Dowex 1 formate
(200-400 mesh) column (0.7
6 cm).
[4-
H]NAD was eluted with 0.6 M formic
acid, and [4-
H]NADP was eluted with 1.2 M formic acid (Hatefi et al., 1980). The contents of the
tubes containing [4-
H]NADP were combined and
lyophilized. [4-
H]NADP was reduced in 0.5 ml of
10 mM sodium phosphate, pH 7.5, containing 4 mM MgCl
, 7.5 mMD-isocitrate, and 120
µg of isocitrate dehydrogenase. The solution was incubated at room
temperature and monitored spectrophotometrically for NADP reduction.
After completion of the reaction (A
/A
= 2.3), the
solution was heated in a boiling water bath for 2 min to inactivate and
precipitate the enzyme. The resulting supernatant obtained after
centrifugation was calibrated spectrophotometrically for
[4-
H]NADPH concentration and then used for
binding studies. The extinction coefficient used for determining the
concentration of NADPH was 6.22 mM
cm
at 340 nm.
Binding Experiments
The centrifugation method of
Howlett et al.(1978) was followed for analyzing the binding of
nucleotides to peptides. Proteins were mixed with different fixed
concentrations of [H]NADPH in 110 µl of 10
mM sodium phosphate (pH 7.0), containing 0.5 mM dithiothreitol and 0.02% potassium cholate. Dextran T40 (2 mg/ml)
was also added to provide density stabilization and prevent convective
stirring of the tube contents during deceleration of the rotor. Samples
were centrifuged at room temperature in a Beckman Airfuge at 30 p.s.i.
Ten µl of the top layer before and after centrifugation was assayed
for radioactivity, and the values were used to calculate total and free
ligand concentrations, respectively. The difference in the
concentration of radioactive ligand between the original solution and
the supernatant after centrifugation was a direct measure of the amount
of bound ligand. The data were treated according to the Scatchard
equation for equilibrium binding (Segel, 1975) as before (Yamaguchi and
Hatefi, 1993).
Gel Electrophoresis
Enzyme samples were incubated
for 1 h at room temperature in 63 mM Tris-HCl, pH 6.8,
containing 2% SDS, 5% -mercaptoethanol, 10% glycerol, and 0.002%
bromphenol blue, and subjected to electrophoresis on 10%
SDS-polyacrylamide slab gels (Laemmli, 1970). Gels were stained with
Coomassie Blue and destained.
Asp-213),
which is located in a 58-residue-long segment of high sequence identity
among the 5 sequenced transhydrogenases from diverse sources (Fig. 1). This 58-residue-long segment immediately precedes the
extramembranous NADP(H) binding domain, and, for the reasons mentioned
in the introduction, makes it a possible candidate for involvement in
energy transduction and the enzyme's proton channel. Hence, it
was of interest to investigate the role of the E. coli transhydrogenase
Asp-213 in transhydrogenation and proton
translocation by site-directed mutagenesis.
), so that no background
wild-type activity, no matter how small, would complicate the
interpretation of results. This was done as described under
``Experimental Procedures,'' and the deletion of
transhydrogenase genes was confirmed by Southern blotting. Then, in
this strain were expressed the wild-type enzyme as well as two mutants
in which
Asp-213 was mutated to Asn (
D213N) and Ile
(
D213I).
Enzymatic Properties of the Mutated Enzymes
Fig. 2shows the transhydrogenase activities of E.
coli membranes containing the expressed wild-type (A) and
the D213N (B) and
D213I (C) mutants. The
transhydrogenase activity of membranes from the TH
strain (D) is also shown. The minimal activity seen in
trace D is probably due to another membrane-bound protein. Mutation of
Asp-213 to Asn and Ile resulted, respectively, in a 72% and 91%
loss of transhydrogenase activity (see Fig. 2legend). These data
indicated that, even though
Asp-213 does not appear to be an
essential residue, its mutation (especially to Ile) results,
nevertheless, in a dramatic loss of transhydrogenase activity. For
further characterization of the mutants, all three expressed enzymes
were purified from the E. coli membranes (Fig. 3).
(
)The purified enzymes were then incorporated into
liposomes and assayed for transhydrogenation-coupled proton
translocation. As seen in Fig. 4, they were all capable of proton
translocation upon initiation of NADPH
AcPyAD
transhydrogenation, and it was apparent that the proton translocation
capability of the
D213I mutant was less than those of the
wild-type and the
D213N mutant enzymes. The initial rates of
9-aminoacridine fluorescence quenching upon initiation of the reaction
by addition of AcPyAD indicated that the rates of proton translocation
by the
D213N and the
D213I mutant enzymes were, respectively,
25% and
9% of the proton translocation rate of the wild-type
enzyme. These values agreed well with the relative transhydrogenation
activities of the purified enzymes as given in the legend to Fig. 3. A more interesting difference between the wild-type and
mutant transhydrogenases was, however, the effect the mutation of
Asp-213 had on the apparent K
values
of the substrates. As seen in , the apparent K
values for NADPH and AcPyAD, as
determined from Lineweaver-Burk double reciprocal plots, were
diminished in the mutant enzymes. This change was particularly
noteworthy for the apparent K
of the
D213I mutant, which had decreased by about 3.5-fold as compared to
that of the wild-type transhydrogenase.
Figure 2:
Comparison of transhydrogenase activities
of E. coli membranes containing expressed wild-type and mutant
enzymes. The NADPH to AcPyAD transhydrogenase activities shown were
each catalyzed by 28 µg of membrane protein. A, wild-type; B, D213N mutant; C,
D213I mutant; D, MC 4100TH
without transformation.
Specific activities of A, B, and C,
calculated from the initial rates of the progress curves shown, are
5.4, 1.52, and 0.48 µmol of AcPyAD reduced (min
mg of
protein)
, respectively. Assay conditions are
described under ``Experimental
Procedures.''
Figure 3:
SDS-polyacrylamide gel electrophoretic
patterns of purified wild-type and mutant transhydrogenases.
SDS-polyacrylamide gel electrophoresis was carried out as described
under ``Experimental Procedures.'' To each lane 3 µg of
purified enzyme were added. A, wild-type enzyme; B,
D213N mutant; C,
D213I mutant. The specific
activities of the purified enzymes A, B, and C were, respectively, 22.5, 5.14, and 2.20 µmol of AcPyAD
reduced (min
mg of
protein)
.
Figure 4:
Proton translocation by proteoliposomes of
wild-type and mutant transhydrogenases. The reaction mixture (2.0 ml)
contained 10 mM Tricine/NaOH, pH 8.0, 2.5 mM MgSO, 0.25 mM dithiothreitol, 0.2 mM
EDTA, 20 µg of liposome-reconstituted transhydrogenase, 1
µM 9-aminoacridine, and 200 µM NADPH. Where
indicated, 400 µM AcPyAD was added to start
transhydrogenation and proton translocation, and 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone was
added to uncouple. The fluorescence changes of 9-aminoacridine were
monitored at 500 nm. Excitation was at 420 nm. A, wild-type
enzyme; B,
D213N mutant; C,
D213I
mutant.
Equilibrium Binding of [
Because the
decreases in the substrate KH]NADPH
to the Wild-type and
D213I Mutant Enzymes
values were
accompanied by V
decreases, it was important to
see whether these K
decreases were a
reflection of k
decreases, or there was in fact
a change in the affinity of the enzyme for substrates. Hence, direct
binding studies were done with the purified wild-type and the
D213I mutant enzymes, using [
H]NADPH.
Binding studies with the
D213N mutant and with AcPyAD were not
done, because the K
changes were not
large enough to be reliably checked by equilibrium binding studies. The
results of equilibrium binding of [
H]NADPH to the
purified wild-type (A) and the
D213I mutant (B)
transhydrogenases are shown in the Scatchard plots of Fig. 5. The
K
values derived from these plots were 19.7
µM for the wild-type transhydrogenase and 6.0 µM for the
D213I mutant enzyme. It may be noted that in Fig. 5the abscissa intercepts (moles of
[
H]NADPH bound per mol of dimeric
transhydrogenase) were n = 0.78 in panel A and n = 0.65 in panel B. Ideally, these values
should be close to 1.0 for a dimeric, half-of-the-sites reactive
enzyme. For the bovine transhydrogenase, we estimated n values
of 1.02 for NADH and 0.92 for NADPH (Yamaguchi and Hatefi, 1993).
However, we have noticed that the purified E. coli transhydrogenase has a greater tendency to aggregate at the
concentrations used for binding studies than the purified bovine
enzyme. We, therefore, repeated the experiments of Fig. 5in the
presence of different low concentrations of lysophosphatidylcholine,
which increase transhydrogenase activity when added to the assay
mixture, but there was no improvement in the values of n.
Figure 5:
Scatchard plots for binding of
[H]NADPH to purified wild-type and
D213I
mutant transhydrogenases. Wild-type enzyme (A, 2.95 mg/ml) and
D213I (B, 2.37 mg/ml) were mixed with different fixed
concentrations of [
H]NADPH (range 1.9-72.7
µM in A and 1.9-53.5 µM in B) in 110 µl of 10 mM sodium phosphate, pH 7.0,
containing 0.02% potassium cholate and dextran T40 (2 mg/ml). The
mixtures were centrifuged at 30 p.s.i. for 45 min. Before and after
centrifugation, 10-µl aliquots of the upper layer of each mixture
were removed, and their radioactivity was measured for calculation of
the molar concentrations of free (L) and enzyme-bound (L) ligand. E is the molar concentration of the
dimeric enzyme. The data were treated according to the Scatchard
equation as described before (Yamaguchi and Hatefi, 1993). The
constants obtained in these experiments were: K
= 19.7 µM, n = 0.78 (A); K
= 6.0
µM, n = 0.65 (B), where n is moles of NADPH bound per mol of dimeric
transhydrogenase.
NADP(H)-dependent NADH
The energy-transducing transhydrogenases of
mitochondria and bacteria do not catalyze transhydrogenation from NADH
to NAD or AcPyAD, but they do catalyze a slow transhydrogenation from
NADPH to NADP or AcPyADP, which was shown in bovine submitochondrial
particles to be energy-linked and accelerated by membrane energization
(Hatefi et al., 1980; Phelps et al., 1980). However,
Fisher and co-workers (Wu et al., 1981) discovered that in
reconstituted proteoliposomes, where the normal NADPH AcPyAD Cyclic
Transhydrogenation
NAD
transhydrogenation becomes inhibited in the absence of an uncoupler to
dissipate the membrane potential, the enzyme does catalyze a rapid and
stoichiometric NADH
AcPyAD transhydrogenation, which requires
NADP(H) and is not coupled to proton translocation. Furthermore, they
showed that whereas the stereospecificity of hydride ion transfer in
the energy-transducing reaction is [4A]NAD(H)
[4B]NADP(H), in the NADP(H)-dependent NADH
NAD (or
AcPyAD) reaction it is 4A
4A (Wu et al., 1981). A
possible explanation offered was that creation of a membrane potential
in proteoliposomes prevents the release of enzyme-bound NADP, which
accepts a hydride ion equivalent from NADH. Then, the NAD so formed
goes off the enzyme and is replaced by AcPyAD, which is reduced by the
enzyme-bound NADPH (Wu et al., 1981; Fisher and Earle, 1982).
Hutton et al.(1994) have shown recently that this cyclic NADP(H)-dependent NADH
AcPyAD transhydrogenation can also
be observed at pH 6.0 and low ionic strength with the
detergent-solubilized enzyme not incorporated in liposomes, apparently
because the enzyme-NADP(H) complex is much more stable under these
conditions. In principle, therefore, the cyclic NADH
AcPyAD
transhydrogenation catalyzed via an enzyme-bound NADP(H) would be
analogous to the [4B]NAD(H)
[4B]NADP(H)
transhydrogenation catalyzed by the so-called BB transhydrogenases.
These enzymes are soluble flavoproteins in which the enzyme-bound
flavin carries out the act of transferring a hydride ion equivalent
from one nicotinamide dinucleotide to another as they bind
interchangeably at the same site (Rydström et al., 1987;
Lee and Ernster, 1989).
NAD
transhydrogenation reaction. In other words, we could ask whether the
inhibited
D213I mutant was capable of catalyzing the
NADP(H)-dependent NADH
AcPyAD cyclic transhydrogenation
reaction. As seen in Fig. 6A, the purified
D213I
mutant catalyzed a rapid cyclic transhydrogenation reaction upon
successive additions of 20 µM NADH to a reaction mixture
containing 0.2 mM each of NADPH and AcPyAD (see also Glavas,
1994). For comparison, the cyclic transhydrogenation reaction catalyzed
by the purified, wild-type enzyme is also shown (Fig. 6B). The data of , derived from
Lineweaver-Burk double-reciprocal plots, show that the rates of the
cyclic transhydrogenation reactions catalyzed by the purified
D213N and
D213I mutated enzymes were somewhat slower at V
than that catalyzed by the wild-type enzyme.
However, comparison of the data of Tables I and II clearly indicate
that, relative to the rates of the wild-type enzyme, the
energy-transducing NADPH
AcPyAD transhydrogenase activity of the
D213I mutant enzyme was inhibited much more than its cyclic NADH
AcPyAD reaction, which is not coupled to proton translocation.
An obvious conclusion, therefore, is that the mutation of
Asp-213
to Ile interferes with the proton translocation capability of the E. coli transhydrogenase, thereby inhibiting the coupled NADPH
AcPyAD transhydrogenation much more than the cyclic NADH
AcPyAD reaction. also shows that in the cyclic
transhydrogenase reaction the apparent S
(the
concentration needed for half-maximal activity) of NADPH decreased in
the mutated enzymes and that in the
D213I mutant it is one order
of magnitude smaller than in the wild-type enzyme. These results are in
agreement with the apparent K
data of and the K
values derived from
the equilibrium binding experiments of Fig. 5. Together, they
suggest that mutations of
Asp-213 to Asn and especially to Ile in
the hydrophobic, membrane-intercalating domain of the enzyme are
communicated to the NADP(H) binding site, altering the enzyme's
affinity for NADPH. The implications of these results on the mechanism
of energy transduction by the transhydrogenase enzyme are discussed
below.
Figure 6:
NADPH-dependent cyclic NADH AcPyAD
transhydrogenation catalyzed by purified wild-type and
D213I
mutated transhydrogenases. The reaction mixtures contained 10 mM MES/KOH, pH 6.0, 10 µg of lysophosphatidylcholine, 0.2 mM NADPH, 0.2 mM AcPyAD, 2.8 µg of purified
D213I (A) or 2.3 µg of purified wild-type (B)
transhydrogenase. After 1 min, 20 µM NADH was added to
start the reaction followed by an additional 20 µM NADH
where indicated by arrows. Reduction of AcPyAD was monitored
at 375 nm.
subunit of nicotinamide nucleotide transhydrogenase
(
Asp-213) to Asn (
D213N) or Ile (
D213I) diminishes in
parallel the hydride ion transfer (NADPH
AcPyAD) and the
transmembrane proton translocation activities of the enzyme. In the
case of the
D213I mutant, these activities, as assayed with the
purified and liposome-reconstituted enzymes, were 90% lower than the
activities of the wild-type transhydrogenase. However, another activity
of the
D213I mutant, namely, the NADP(H)-dependent cyclic NADH
AcPyAD transhydrogenation, which is not coupled to proton
translocation, was only about 35% inhibited. As seen in Fig. 1,
Asp-213 is located in a hydrophobic stretch of 58 highly conserved
amino acid residues, which is immediately adjacent to the COOH-terminal
extramembranous domain of the
subunit that binds NADP(H). In the
hydrophobic domain of the
subunit, there is a second, much
shorter stretch (19 residues) of high sequence conservation (Fig. 1). This segment contains a conserved His (
His-91)
whose mutation to Ser, Thr, or Cys also results in
90% loss of
NADPH
AcPyAD hydride ion transfer and coupled proton
translocation activities (Holmberg et al., 1994). Because such
regions of high sequence identity are rare in the hydrophobic domains
of functionally related proteins, it is possible that in the folded
structure of the transhydrogenase these regions of high sequence
identity are in close apposition and are together involved as
membrane-intercalated helices in the proton translocation function of
the enzyme. Whether
His-91 and
Asp-213 are within these
membrane-intercalated helices or in the extramembranous loops that
connect the helices is not known. Nor do the catalytic properties of
the mutant enzymes cited above indicate whether these amino acid
residues play a chemical or a structural role in the coupled reactions
of the transhydrogenase. Such distinctions are difficult to make,
especially when one considers the recent results of Sonar et
al.(1994) on bacteriorhodopsin. These authors have shown that in a
Y57D mutant of bacteriorhodopsin the deprotonation of the retinal
Schiff base and formation of the M intermediate is blocked.
Nevertheless, the Y57D mutant is capable of appreciable proton
translocation activity, apparently via a redirected proton pathway.
Therefore, in the E. coli transhydrogenase, the incomplete loss of proton translocation activity upon mutations of
His-91 and
Asp-213 does not necessarily mean that one or both
of these residues are not directly involved in proton translocation in
the wild-type enzyme.
AcPyAD transhydrogenation and coupled
proton translocation activities, the
D213I mutation lowers the
apparent K
by 3.5-fold in the above
reaction (assayed at pH 7.0) and by 10-fold in the cyclic NADH
AcPyAD transhydrogenation reaction (assayed at pH 6.0 and low ionic
strength). In agreement with these results, it was shown by equilibrium
binding experiments (at pH 7.0) that in the
D213I mutant enzyme K
is
3-fold lower than in the wild-type
transhydrogenase. These results indicate, therefore, that
Asp-213
or a mutation of this residue influences the affinity of the nearby
NADP(H) binding site for its substrate. We have discussed elsewhere
that the energetically uphill (inside-to-outside) proton translocation
that is coupled to NADPH
NAD transhydrogenation (reversal of
Equation 1) is driven by the difference in the binding energies of the
reactants (NADPH and NAD) and the products (NADP and NADH). This means
that the enzyme must undergo a conformation change coupled to hydride
ion transfer from NADPH to NAD. Because substrate binding by the bovine
transhydrogenase is random, we were able to show that the bindings of
NADPH and NADP, but not of NADH and NAD, change the conformation of the
enzyme in different ways. We, therefore, proposed that the difference
in the binding energies of NADPH and NADP is the principal force that
via coupled conformation changes of the protein results in vectorial
proton release and uptake across the membrane (for elegant discussions
of the utilization of binding energy to drive coupled vectorial
processes, including proton translocation, see Jencks(1975, 1989)).
Accordingly, if we now assume that the hydrophobic stretches of high
sequence identity depicted in Fig. 1are involved in the coupled
conformation changes of the transhydrogenase, we could then rationalize
why a point mutation in these hydrophobic stretches might alter the
affinity of the enzyme for NADPH. In other words, it is possible that
the
D213I mutation causes a structural change in the hydrophobic
domain of the
subunit that has a reciprocal effect on the binding
energy of NADPH. This structural change could restrict proton
translocation and inhibit the coupled transhydrogenation reaction.
D213I mutation, is that the enzyme's cyclic NADH
AcPyAD transhydrogenase activity is much less inhibited than its
normal, energy-coupled NADPH
AcPyAD transhydrogenation reaction.
The inhibition of the latter reaction, as discussed above, is
consistent with the possibility that the
D213I mutation restricts
proton translocation and as a consequence inhibits the coupled NADPH
AcPyAD transhydrogenation. However, this does not explain why
the cyclic NADH
AcPyAD reaction should not be equally inhibited
as well. The explanation, we feel, lies in the difference between the
normal and the cyclic transhydrogenation reactions, more precisely
between the role of NADP(H) in each. In the energy-coupled NADPH
NAD transhydrogenation reaction, enzyme-bound NADPH and NADP are in
equilibrium with their counterparts in the medium. As a result, the
enzyme experiences their different binding energies with each turnover
and undergoes the conformation changes that drive proton translocation.
By contrast, the cyclic NADH
NAD transhydrogenation occurs while
NADP(H) is enzyme-bound. Hence, the enzyme does not experience the
different binding energies of NADPH and NADP and does not undergo the
conformation changes that drive proton translocation. This important
difference can, therefore, explain why the cyclic NADH
AcPyAD
transhydrogenation is not coupled to proton translocation and why
restriction of the latter process by the
D213I mutation does not
have a comparable inhibitory effect on the cyclic transhydrogenation
reaction.
Table: Kinetic parameters of wild-type and mutant
enzymes for the NADPH AcPyAD transhydrogenation reaction
and V
values were derived from Lineweaver-Burk double reciprocal plots.
Table: Effect of NADPH concentration on the
cyclic NADH AcPyAD transhydrogenation catalyzed by the purified
wild-type and mutant enzymes
(NADPH
concentration required for half-maximal activity) and V
values were derived from Lineweaver-Burk
double reciprocal plots.
D213N and
D213I mutations.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.