(Received for publication, December 1, 1995; and in revised form, February 5, 1996)
From the
Transhydrogenase catalyzes the reduction of NADP by NADH coupled to the translocation of protons across a
membrane. The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding
domain (called Th
) exists as a separate polypeptide.
Th
was expressed in Escherichia coli and purified.
The binding of nucleotide substrates and analogues to Th
was examined by one-dimensional proton nuclear magnetic resonance
(NMR) spectroscopy and by measuring the quenching of fluorescence of
its lone Trp residue. NADH and reduced acetylpyridine adenine
dinucleotide bound tightly to Th
, whereas
NAD
, oxidized acetylpyridine adenine dinucleotide,
deamino-NADH, 5`-AMP and adenosine bound less tightly. Reduced
nicotinamide mononucleotide, NADPH and 2`-AMP bound only very weakly to
Th
. The difference in the binding affinity between NADH and
NAD
indicates that there may be an energy requirement
for the transfer of reducing equivalents into this site in the complete
enzyme under physiological conditions. Earlier results had revealed a
mobile loop at the surface of Th
(Diggle, C., Cotton, N. P.
J., Grimley, R. L., Quirk, P. G., Thomas, C. M., and Jackson, J. B.
(1995) Eur. J. Biochem. 232, 315-326); the loop loses
mobility when Th
binds nucleotide; the reaction involves
two steps. This was more clearly evident, even for tight-binding
nucleotides, when experiments were carried out at higher temperatures
(37 °C), where the resonances of the mobile loop were substantially
narrower. The binding of adenosine was sufficient to initiate loop
closure; the presence of a reduced nicotinamide moiety in the
dinucleotide apparently serves to tighten the binding. Two-dimensional
H NMR spectroscopy of the Th
-5`-AMP complex
revealed nuclear Overhauser effect interactions between protons of
amino acid residues in the mobile loop (including those in a Tyr
residue) and the nucleotide. This suggests that, in the complex, the
loop has closed down to within 0.5 nm of the nucleotide.
Transhydrogenase couples the transfer of reducing equivalents between NAD(H) and NADP(H) to translocation of protons across a membrane(1, 2, 3) .
The enzyme is located in the cytoplasmic membrane of bacteria, where it is responsible for NADPH production during biosynthesis, and in the inner mitochondrial membrane, where it may serve in regulation of the tricarboxylic acid cycle by the protonmotive force(4) .
Transhydrogenase has separate catalytic binding sites for NAD(H) and
for NADP(H)(5, 6, 7, 8) . These
reside in relatively hydrophilic domains of the protein that protrude
from the cytoplasmic side of the membrane (in bacteria). The NAD(H)
site resides in domain I and the NADP(H) site in domain
III(9) . The hydrophobic domain II spans the membrane. Several
primary sequences of transhydrogenase are known. All are similar,
although the polypeptide composition is variable (see (3) and (10) ). Uniquely, in Rhodospirillum rubrum transhydrogenase the NAD(H)-binding domain I exists as a separate
polypeptide (called
Th)(
)(10, 11, 12) .
This polypeptide, in dimeric form, can be dissociated from the domain
II/III components of the enzyme. Separated Th
and the
domain II/III proteins lack transhydrogenation activity but
reconstitution leads to full recovery.
Recombinant Th from R. rubrum was expressed at high levels in Escherichia coli(14) . The purified recombinant
protein completely restored transhydrogenation activity to membranes of R. rubrum that had been washed to remove native domain I
polypeptide. The characteristics of NADH binding to Th
were
investigated by monitoring fluorescence quenching of a sole Trp residue
at position 72 in the recombinant protein(14) . From an
analysis of the one-dimensional
H NMR spectrum of
Th
, it was proposed that a region, which straddles sites
that are highly sensitive to cleavage by proteases
(Lys
-Thr
and
Lys
-Glu
), has a significantly greater
segmental flexibility than the remainder of the molecule(15) .
This segment might be a mobile loop emanating from the surface of the
protein. Some of the sharper resonances in the NMR spectrum were
provisionally assigned to specific amino acids.
The well defined H resonances attributed to amino acid residues within the
mobile loop were considerably broadened and some were slightly shifted
when either NAD
or NADH were added to recombinant
Th
(15) . This indicates that nucleotide binding
causes a loss of loop mobility, and alters the rates of chemical
exchange processes between conformations as the protons sample a range
of environments on the NMR time scale. Titration with NAD
revealed a two-step binding reaction. At low concentrations of
nucleotide predominantly one resonance, the upfield Met at 1.97 ppm,
was broadened. At higher concentrations, other mobile loop resonances
also began to broaden. In titrations with NADH, relatively low
nucleotide concentrations led to broadening of the mobile loop
resonances. The resulting NMR spectrum was observed in the presence of
higher concentrations of NAD
.
In this report we
describe the binding characteristics of NAD(H) analogues to Th with a view to establishing nucleotide specificity and defining
the conformational dynamics of the mobile loop during the
nucleotide-binding reaction. Two-dimensional proton NMR spectra of the
Th
-5`-AMP complex reveal interactions between nucleotide
and protein.
Preparation of the strain of E. coli bearing the
recombinant Th gene from R. rubrum, growth and
induction of bacteria, and purification of the protein were carried out
as described(14) . The procedure for preparing R. rubrum membrane vesicles depleted of native Th
was
described(11) .
The factor used to correct protein
concentrations in colorimetric assays (14) was found to be
unreliable because traces of glycerol that were present, even after
dialysis, resulted in an underestimate in the amino acid analysis of
samples hydrolyzed with HCl. To ensure that protein estimations were
correct, Th (more than 95% pure according to
SDS-polyacrylamide gel electrophoresis; (14) ) was estimated
using two procedures, the microtannin assay (16) and the
bicinchoninic acid assay(17) , which rely on different
reactions of the protein, using bovine serum albumin as standard. The
results were similar within 2%. In turn these concentrations were
similar within 25% and 5%, respectively, to concentrations of solutions
of native protein, and of protein denatured in 8 M urea,
calculated from the absorbance at 280 nm, using an extinction
coefficient predicted from the content of aromatic amino acids in
Th
. Routine measurements of the protein were subsequently
estimated by the microtannin assay.
Fluorescence spectroscopy was
performed with a Spex Fluoromax. Fluorescence signals were recorded at
25 °C in a stirred 1 1-cm cuvette using excitation and
emission wavelengths of 280 nm and 310 nm, respectively. Slit widths
were 1 mm. In titrations with nucleotides, inner filtering effects were
compensated as described(14) .
Samples of Th for
NMR spectroscopy were prepared as described(15) .
H
NMR spectra were recorded on a Bruker AMX500 spectrometer.
One-dimensional pulse and collect spectra were acquired using a 10-ppm
sweep width and comprised 256, 512, or 1024 transients of 16,000 data
points with a total acquisition time of 45 min (or less; see figure
legends). The temperature was 20 °C unless otherwise indicated.
Spectral data were processed, prior to Fourier transformation, by
application of an exponential multiplication factor corresponding to a
line broadening of 1 Hz. In two-dimensional nuclear Overhauser effect
spectroscopy (NOESY) experiments, 2000 data points were collected for
224 rows, each of 128 transients. Total acquisition time was 17 h.
Figure 1:
Representative one-dimensional H NMR spectra recorded during a titration of Th
with AcPdAD
. A, NMR spectrum of 200
µM Th
(512 transients), in 10 mM [
H]Tris-Cl, p
H 7.6, 10 mM (NH
)
SO
, 0.5 mM dithiothreitol in
H
O, at 20 °C, in the
absence of nucleotides (note that the very sharp resonances at around
1.2-1.5 ppm are impurities); B, plus 50 µM AcPdAD
; C, plus 400 µM AcPdAD
. The p
H was checked after
nucleotide addition and, if necessary, adjusted to 7.6 with
NaO
H.
The reduced nucleotide, AcPdADH, binds to
Th and produces conformational changes in the mobile loop
similar to those observed during NADH binding. 1) Addition of AcPdADH
to a solution of Th
led to quenching of Trp
fluorescence (Fig. 2). For similar concentrations of
nucleotide, quenching was less extensive with AcPdADH than with NADH.
Because of limitations imposed by inner-filtering effects of the
nucleotides(14) , the K
of the analogue
was difficult to determine with precision, but assuming the extent of
fluorescence quenching at saturating AcPdADH was similar to that with
NADH, the K
was approximately 40 µM (cf. approximately 20 µM for NADH; (14) ). 2) Titration of Th
with AcPdADH led to
changes in the NMR spectrum similar to those observed in NADH
titrations (Fig. 3; compare (15) ). Thus, low
concentrations of nucleotide (30-100 µM for 122
µM protein) led to extensive broadening of resonances
assigned to the mobile loop, i.e. those at 1.22, 1.43, 1.97,
2.06, 2.28, 6.82, 7.12, and 7.33 ppm (assigned as above). Also similar
to NADH titration spectra, resonances of nucleotide protons were
undetectable (due to line broadening) until high concentrations
(>400 µM for 122 µM protein) of AcPdADH
were added.
Figure 2:
The quenching of fluorescence of the lone
Trp of Th by nucleotides. Experiments were performed with
0.6 µM Th
in a medium containing 10 mM Tris-HCl, pH8.0, 10 mM (NH
)
SO
, 1 mM dithiothreitol. The data are corrected for inner filtering effects
(see ``Materials and Methods'').
, NADH;
,
AcPdADH;
, deamino-NADH;
,
NAD
.
Figure 3:
Representative one-dimensional H NMR spectra recorded during a titration of Th
with AcPdADH. For conditions, see Fig. 1. A, NMR
spectrum of 122 µM Th
(512 transients), in the
absence of nucleotides; B, plus 30 µM AcPdADH.
We speculated(15) , although it was not clearly
evident from NMR spectra, that NADH as well as NAD might bind to Th
in a two-step process. Detection of
the intermediate state might be difficult at 20 °C because of
differences in rates of chemical exchange between reaction
intermediates that involve reduced nucleotide. Experiments at 37 °C
indicate that for both NADH and AcPdADH a two-step reaction can indeed
be resolved. Reconstitution experiments with depleted membrane vesicles
showed that Th
retains activity when incubated at 37 °C
for >24 h. The NMR spectrum of Th
at 37 °C in the
absence of nucleotides is shown in Fig. 4A. The
resonances were much sharper than those at 20 °C (compare Fig. 1and Fig. 3), indicating that at higher temperatures
the loop takes on even greater segmental mobility. Titration of
Th
at 37 °C with either NADH (representative data in Fig. 4, B and C) or AcPdADH (data not shown)
revealed the two-step binding reaction. At low concentrations of
reduced nucleotide (20 µM; Fig. 4B), the
MetA resonance at 1.97 ppm was shifted (0.02 ppm) upfield and was
broadened more extensively than those attributable to Thr, Ala, Tyr,
etc. in the mobile loop. At slightly higher concentrations of NADH (100
µM; Fig. 4C) or AcPdADH (data not shown),
other loop resonances did become broadened.
Figure 4:
Representative one-dimensional H NMR spectra recorded at 37
C during a
titration of Th
with NADH. For conditions, see Fig. 1, except that the temperature was 37 °C. A,
NMR spectrum of 240 µM Th
(256 transients), in
the absence of nucleotides; B, plus 20 µM NADH; C, plus 100 µM NADH.
Deamino-NADH is modified
in the adenine part and not in the nicotinamide part with respect to
NADH. It had a small quenching effect on fluorescence from Trp of Th
(Fig. 2), but the K
was too large to measure. The weak binding of this analogue to
Th
was also reflected by NMR data. Thus, features observed
in NADH titrations were also seen with deamino-NADH, but at higher
concentrations (about 2-fold for equivalent protein concentrations, not
shown). The two-step reaction was evident, the MetA resonance
broadening at lower nucleotide concentrations than other mobile loop
resonances.
5`-AMP and adenosine are weak inhibitors of
transhydrogenase(19) . Inhibition with 5`-AMP is competitive
with respect to AcPdAD and mixed with respect to
NADPH(5, 6, 8) . Remarkably, the effects of
5`-AMP and adenosine on the NMR spectrum of Th
were
qualitatively very similar to those of NAD
and
AcPdAD
; low concentrations specifically broadened and
shifted slightly upfield the MetA resonance, and higher concentrations
led to broadening of the other mobile loop resonances (data not shown).
The nucleotide resonances became evident as narrow bands in the
spectrum even at quite low concentrations of 5`-AMP and adenosine (e.g. 150 µM 5`-AMP for 200 µM protein).
Reduced nicotinamide mononucleotide (NMNH)
corresponds to the complementary half of NADH to 5`-AMP. It did not
quench fluorescence of Trp, and, up to 1 mM,
either on its own or in combination with 400 µM 5`-AMP,
NMNH had no effect on the NMR spectrum of Th
(data not
shown).
In contrast to NADH, NADPH up to about 50 µM did not quench the fluorescence from Trp of Th
and addition of NADPH up to 200 µM had little effect
on the NMR spectrum (data not shown). High concentrations of NADPH
(around 1 mM) led to slight broadening. Again in contrast to
NADH, the nucleotide resonances became evident as sharp bands, even at
at low concentrations of NADPH (50 µM), during the
titration with Th
(208 µM). Together, these
data indicate that NADPH can bind into the NADH site, at best with only
very low affinity, and the possibility is not ruled out that other
contaminating nucleotide(s) in the NADPH solution cause the protein
resonance broadening.
2`-AMP is an inhibitor of
transhydrogenase(8, 5, 6, 19) ,
competitive with respect to NADPH and mixed with respect to
AcPdAD. 2`-AMP had no effect on the NMR spectrum of
Th
, and nucleotide resonances were detectable at very low
nucleotide concentrations (data not shown).
Figure 5:
The pH dependence of NADH binding to
Th. Experiments were performed as in Fig. 2, except
that instead of Tris-HCl, the medium contained 20 mM Ches, 20
mM Tricine, 20 mM Mops with the pH adjusted to that
shown in the figure with NaOH. The concentration of Th
was
1.0 µM. K
values were
calculated from double-reciprocal plots of the corrected
data.
The one-dimensional proton
NMR spectrum of Th at pH6.3 in cacodylate buffer (data not
shown) was similar to that recorded in Tris-Cl buffer at pH7.6. The
unassigned resonances between 7.6 and 7.9 ppm were slightly broader at
the lower pH, which might indicate that they arise from His residues,
but other resonances displayed a similar chemical shift and linewidth.
Consistent with the lack of effect of pH on K
, NMR
spectra recorded during an NADH titration of Th
at pH6.3
(data not shown) were similar to those at pH7.6(15) .
Th bound to NADH was not suitable for NOESY experiments because of
the extreme broadening of the resonances from both molecules at the
high concentrations of NADH required(15) . The analogue 5`-AMP,
with a lower binding affinity, was preferred. Experiments were
performed on a mixture of Th
and 5`-AMP, control
experiments on Th
and 5`-AMP alone. Very few NOE
interactions were detected in Th
alone; interesting
exceptions were from Tyr
ring protons (see (22) for confirmation of this assignment) to a resonance at
4.52 ppm, likely to be the Tyr
C
H (Fig. 6A). Additional cross-peaks were detected in the
Th
/5`-AMP mixture, with the H8, H2, H1`, H4`, H5`, and H5"
protons of 5`-AMP all showing interactions with protons of Th
(Fig. 6). Although absolute assignments are not possible,
the H8 proton in particular appeared to interact with C
H of
Tyr
, a further C
H (possibly Lys or Ala), and side
chain methylene and methyl groups. The Tyr
ring
proton-C
H interaction at 4.52 ppm in the absence of nucleotide
(see above) was shifted to 4.58 ppm, and decreased in intensity, in the
presence of 5`-AMP.
Figure 6:
Two-dimensional H NMR of
Th
. Experiments were performed as described in Fig. 1and under ``Materials and Methods.'' A,
200 µM Th
alone; B, plus 600
µM 5`-AMP. Controls with 600 µM 5`-AMP alone
are not shown.
The reduced nucleotides, NADH and AcPdADH, bind more tightly
to Th than do the oxidized nucleotides, NAD
and AcPdAD
. (a) Low concentrations of
NADH and AcPdADH quenched the fluorescence of Trp
of the
protein, whereas NAD
and AcPdAD
up to
50 µM did not; (b) equilibrium dialysis
measurements (
)give K
values for NADH
consistent with those measured from Trp
fluorescence
quenching, and indicate higher values for NAD
; (c) in NMR spectra recorded during titrations, the resonances
of NADH and AcPdADH were broad until quite high concentrations were
added to Th
, but those of NAD
and
AcPdAD
appeared as sharp bands even at quite low
concentrations; and (d) resonances attributable to the mobile
loop of Th
were broadened at relatively low concentrations
of NADH and AcPdADH, but only at high concentrations of NAD
and AcPdAD
. In principle, NMR spectroscopy can
be used to measure binding constants, but the mobile loop resonances of
Th
are insufficiently resolved to allow the detailed
analysis of linewidth and lineshape as a function of nucleotide
concentration required for determination of the type of exchange and
therefore accurate values of K
. Nevertheless, the
dependence of the amplitude of the reasonably resolved resonances (e.g. Thr, Ala, and Tyr) on nucleotide concentration should
give a comparative indication of binding affinities: there could be a
5-10-fold difference in the K
values
(K
> K
and
K
> K
).
Supporting the conclusions from kinetic
analyses(4, 5, 6, 7, 8) ,
the experiments with analogues indicate: (a) the adenosine
moiety is crucial in the binding reaction and (b) the reduced
nicotinamide moiety increases the binding affinity when it is part of
the NADH molecule, but that, as a free entity, NMNH cannot occupy the
binding site. The oxidized nicotinamide moiety seems barely to
contribute to the affinity since, on the basis of the NMR spectra,
NAD appeared to bind only a little more tightly than
5`-AMP and adenosine. The importance of the adenosine part is also
highlighted by the observation that deamino-NADH bound more weakly than
NADH. Different patterns of inhibition of the transhydrogenation
reaction by 5`-AMP (an NAD(H) analogue) and 2`-AMP (an NADP(H)
analogue) were instrumental in the development of the now established
concept that the complete enzyme has separate sites for NAD(H) and
NADP(H)(4, 5, 6, 7, 8, 19) .
The observation from NMR spectra that, in contrast to 5`-AMP, 2`-AMP
fails to bind to Th
is a complementary view of those
inhibition patterns. That adenosine binds to Th
with the
same order of affinity as 5`-AMP indicates that lack of the
5`-phosphate is not critical for binding, but comparison with the
2`-AMP results shows that presence of the 2`-phosphate blocks
nucleotide binding at this site.
The finding that the binding
affinity of Th for NADH is substantially greater than that
for NAD
may be considered in the context of the
operation of transhydrogenase under physiological conditions. If the
enzyme operates in the direction of reduction of NADP
by NADH, then it must bind NADH from, and release NAD
to, an environment (the mitochondrial matrix or the bacterial
cytoplasm) in which free NAD(H) is predominantly oxidized. Thus, tight
binding of NADH to transhydrogenase, and weak binding of
NAD
, favor operation in this direction. The
observation that the binding constants for NAD
and
NADH are different in Th
indicates that they are
intrinsically different (K
>
K
) also in the complete enzyme. If there is
such a difference then, during turnover, energy will be required to
populate the E
NAD
NADPH state from E
NADH
NADP
; this must derive
exclusively from the protonmotive force (
p), since
G for the overall chemical reaction under physiological
conditions is positive. There is evidence that primary energy coupling
in transhydrogenase is centered on the NADP(H) binding reaction
within domains II and III; proton binding and release components of the
proton translocation reaction are coupled directly to NADP
binding and NADPH release, respectively (23, 24, 25) . Thus energy required to drive
the unfavorable hydride transfer reaction (E
NADH
NADP
E
NAD
NADPH) must be derived from
those primary events. It is proposed that conformational changes driven
by the protolytic reactions that accompany NADP(H) binding and release promote the hydride transfer from NADH to
NADP
, either by making the NADH more reducing or by
making the NADP
more oxidizing (compare(26) ).
In structural terms, interactions between protein side chains and
nucleotide in the binding site in domain I must be relatively favorable
for NADH and unfavorable for NAD
;
p must
drive hydride transfer against these unfavorable structural changes
before the protein can release NAD
. The
G required to maintain the NAD
/NADH ratio on the
enzyme against a 10-fold increase in K
(K
= 20 µM;
K
200 µM; see above) is
approximately 5.7 kJ
mol
. The energy available
from a
p of 200 mV is 19.3 kJ
mol
(assuming one proton translocated per H
transferred between nucleotides; (27) ). The balance of
13.6 kJ
mol
is available at the primary
energy-coupling reaction to generate the change in binding constants
for NADP(H) that are associated with protonation/deprotonation; this
G is sufficient to increase
K
/K
by 230-fold.
K values of transhydrogenase for NADH and
NAD
are affected by membrane energization, and this
was interpreted as evidence for a different model to the one above; it
was thought that changes in K
might reflect
changes in affinity for NADH and NAD
during
energization(20, 21) . However, caution is necessary
when interpreting changes in K
for complex
reactions whose mechanism is not understood. Thus, even within the
framework of our model, in which there are no
p-dependent
changes in the affinity for NAD
or NADH, changes in
the K
for these nucleotides are still
expected(24) . Evidence that binding of NADH is not coupled to
proton binding or release in the domain I protein of transhydrogenase
is given under ``Results''; the K
of
Th
for NADH (measured by quenching of Trp
fluorescence) was independent of pH from 6.0 to 9.0 (Fig. 5), and there were no differences in NMR spectra recorded
during NADH titrations at pH 6.3 and 7.6. Energy-linked changes in the
binding affinity of either NAD
or NADH during turnover
cannot be ruled out, but (a) it is unnecessary to invoke them
to describe known properties of the enzyme, and (b) Th
is a very stable, water-soluble protein, which readily restores
transhydrogenation activity to depleted membranes, and thus it is
reasonable to assume that its properties reliably reflect those of
domain I in the complete enzyme.
The question arises as to the role
of the mobile loop during catalytic turnover. An important observation (Fig. 6) of NOE interactions between a Tyr residue in Th and 5`-AMP bound within the NAD(H) site indicates that the
nucleotide and the amino acid residue are in close proximity. It is
shown by mutagenesis that this is Tyr
in
Th
(22) . Thus, the earlier conclusion, that the
loop loses segmental mobility when Th
binds
NAD(H)(15) , can now be refined; upon binding nucleotide, the
loop closes down on the surface of the protein such that some loop
residues are very close (<0.5 nm) to the nucleotide.
The
one-dimensional NMR data show a similar pattern of behavior for
different nucleotides that bind to the NAD(H) site of Th.
Evidently, this behavior is not triggered by the reduced nicotinamide
moiety, which in this context only increases the binding affinity of
the nucleotide, but by the adenosine group. In all cases the furthest
upfield of the proton resonances attributed to Met residues (MetA) is
especially sensitive to nucleotide binding; the resonance is broadened
at lower concentrations of nucleotide than are required to broaden
other loop resonances. The identity of this residue is not known, but
Met
is a good candidate(15) . It is proposed
that, following binding of nucleotide, the protein adopts a
conformation in which the chemical environment of protons in MetA has
changed; for example the residue might lose mobility, resulting in a
broader NMR signal. It is clear (e.g.. Fig. 4),
particularly for NADH and AcPdADH, at 37 °C, that broadening of the
MetA resonance occurs at nucleotide concentrations considerably lower
than the protein concentration. Therefore, the exchange rate between
the two conformational states is probably intermediate/fast for NADH on
the NMR timescale and fast for NAD
. The state in which
the MetA resonance is specifically broadened probably represents an
intermediate on the way to the conformation in which other resonances
(the 2.06 ppm MetC resonance, and the Thr, Ala, Tyr, Phe resonances,
etc.) are also broadened (15) ie. in which the loop
has closed down on the NAD(H)-binding site. The NMR spectrum of this
conformation is evidently more related to its solution concentration,
and hence exchange with the intermediate state is relatively slow. The
binding process can be summarized (15) by the following
reaction.
This is consistent with the inverse relationship between the
appearance of nucleotide resonances during titration of Th with different nucleotides, and the broadening of loop
resonances. For those that bind tightly (NADH and AcPdADH), nucleotide
resonances are not resolved from base-line noise (i.e. they
remain extensively broadened) until they are present at concentrations
somewhat in excess of the protein, whereas loop resonances are
broadened at very low concentrations of the nucleotide. For those which
bind weakly (NAD
and 5`-AMP) nucleotide resonances
emerge from base-line noise as sharp bands even at quite low nucleotide
concentrations; higher concentrations are needed to cause broadening of
loop resonances. In titrations with NADPH and 2`-AMP (which do not bind
significantly), their resonances were evident as narrow bands even at
very low nucleotide concentrations. The broadening of nucleotide
resonances probably results from decreased mobility upon binding to the
protein and, in principle, may occur in both Th
-NAD(H) and
Th
-NAD(H). The less extensive broadening of the
more weakly-bound NAD
and AcPdAD
arises as a result of faster exchange, and/or because the
solution concentration of the unbound nucleotide, for a given total
concentration, is higher.
Fjellstrom et al.(28) developed a model of part of domain I of
transhydrogenase, based on sequence similarities with known
three-dimensional structures of NAD(H)-binding domains of
dehydrogenases. They describe their model with a bound nucleotide, and
thus it should be compared with the situation (see above) in which the
NMR-visible mobile loop has closed down on the NAD(H) binding site.
Unfortunately, no such feature is evident in the predicted structure of
Fjellstrom et al. The segment of polypeptide chain that we
define as the ``mobile loop'' is shown as a short loop at the
C terminus of a predicted -strand (
C) together with the N
terminus of a predicted
-helix (
D)(28) . This feature
is envisaged as pointing away from the bound nucleotide. In
contradiction with NMR data, the E. coli equivalent of
Tyr
in the model is situated >0.5 nm from the bound
nucleotide. The model of Fjellstrom et al. retains attractive
features. Perhaps the choice of dihydropteridine reductase as template
for
D, based only on its length and some homologies in the
subsequent
-strand (28) was inappropriate. It is
conceivable that this segment is unique to transhydrogenase, which
unlike the templates used by Fjellstrom et al. is a
transmembrane ion pump.