(Received for publication, December 1, 1995; and in revised form, February 5, 1996)
From the
The Tyr residue in the mobile loop region of the soluble, domain
I polypeptide (called Th) of the proton-translocating
transhydrogenase from Rhodospirillum rubrum has been
substituted by Asn and by Phe. The recombinant proteins were expressed
at high levels in Escherichia coli and purified to
homogeneity. The two well defined resonances at 6.82 and 7.12ppm,
observed in the one-dimensional proton NMR spectrum of wild-type
protein, and previously attributed to the Tyr residue, were absent in
both mutants. In the Tyr
Phe mutant
Th
, they were replaced by two new resonances at 7.26 and
7.33 ppm, characteristic of a Phe residue. In both mutants, narrow
resonances attributable to Met residues (and in the Tyr
Phe mutant, resonances attributable to Ala residues) were
shifted relative to the wild type, but other features in the NMR
spectra were unaffected. The conformational dynamics of the mobile loop
closure in response to nucleotide binding by the protein were altered
in the two mutants. The fluorescence emission from Trp
was
unaffected by both Tyr substitutions, and the fluorescence was still
quenched by NADH. The mutant Th
proteins bound to
chromatophore membranes depleted of their native Th
with
undiminished affinity. In these reconstituted systems, the K
values for thio-NADP
and NADH, during light-driven transhydrogenation, were similar to
those of wild-type, but the k
values were
decreased about 2-fold. In reverse transhydrogenation, the K
values for NADPH were slightly
decreased in the mutants relative to wild-type, but those for acetyl
pyridine adenine dinucleotide were increased about 10- and 13-fold,
respectively, and the k
values were decreased
about 2- and 5-fold, respectively, in the Tyr
Phe
and Tyr
Asn mutants. It is concluded that
Tyr
may contribute to the process of nucleotide binding
and that substitution of this residue prevents proper functioning of
the mobile loop in catalysis.
In animal mitochondria and bacteria, transhydrogenase is driven
in the direction of NADP reduction by the protonmotive
force generated through the action of respiratory (or photosynthetic)
electron transport chains.
Uniquely in Rhodospirillum rubrum, the NAD(H)-binding
domain I of transhydrogenase exists as a separate
polypeptide(1, 2, 3) . This polypeptide can
be expressed in large quantities in Escherichia coli and
purified as a water-soluble protein (4) . Like the native
protein (called Th), (
)the recombinant form is
dimeric and can restore transhydrogenation activity to everted
membranes (chromatophores) of R. rubrum, which have been
washed to remove native Th
. Th
binds NADH with
a K
of about 20
µM(4) .
Domain I of transhydrogenase has a
mobile loop straddling protease-sensitive sites
(Lys-Thr
and Lys
-Glu
in R. rubrum Th
). It is detectable by NMR,
and its conformation is altered when the protein binds
nucleotides(5) . A Gly-Tyr-Ala motif (residues 234-236 in
the R. rubrum protein) in this region is conserved in all
known transhydrogenase sequences. It was proposed that 3,5 and 2,6 ring
protons of Tyr in the motif give rise to resonances at 6.82 and 7.12
ppm, respectively, in the NMR spectrum of R. rubrum Th
, and equivalent resonances in the spectrum of E. coli domain I protein(5) . Here we test this
hypothesis by examining mutants of R. rubrum Th
in
which Tyr
has been substituted by Asn or Phe. Because the
residue is conserved, and approaches nucleotide to within 0.5 nm in
domain I-AMP complex(13) , Tyr
might have a role
in catalysis. We examine the effect of Tyr
Asn and
Tyr
Phe substitutions in Th
on the
binding of NADH, as judged by quenching of fluorescence of the lone Trp
residue of the protein, on conformational dynamics of the loop during
the binding of NAD(H) and analogues as determined by NMR, and on
catalytic activity and Michaelis constants of mutant protein in forward
(energy-linked) and reverse transhydrogenation after reconstitution
with depleted R. rubrum membranes bearing domain II/III
proteins of the enzyme. The results are compared with those in which
the effect on reverse transhydrogenation activity of mutating the
equivalent residue in E. coli enzyme was measured in membrane
fractions(6) .
Mutagenesis was carried out by the gapped duplex method(8) . The 700-base pair EcoRI-HindIII fragment of pCD1 (4) was cloned into the EcoRI-HindIII site of pMa (8) . Following mutagenesis, the EcoRIHindIII fragment was cloned back into pCD1 and transformed into E. coli C600. The fragment was then sequenced to check for errors; none were found.
Everted membrane vesicles (chromatophores) from
phototrophically-grown cells of R. rubrum were prepared and
depleted of their native Th as
described(1, 10) . Chromatophores of strains that
overexpress transhydrogenase (11) were depleted of Th
by washing 3-4 times in 2 M NaCl, 10 mM Tris-HCl, pH 8.0.
Protein was estimated by the microtannin assay(12) , without using the correction procedure determined(4) , see(13) . Bacteriochlorophyll was measured using the in vivo extinction coefficient(14) . SDS-polyacrylamide gel electrophoresis was carried out as described (15) .
NMR spectra of purified wild-type and mutant Th are shown in Fig. 1. The general characteristics of the
spectra, including broad humps of aromatic and methyl protons from
9-6 ppm and 2.3-0.3 ppm, respectively, were similar,
indicating that the overall fold of the mutant proteins was unchanged
by the amino acid substitutions. Like wild-type Th
, those
of the mutants' spectra displayed a number of narrower resonances
superimposed upon the broad methyl and aromatic proton absorptions.
Some or all of these resonances probably derive from amino acids in a
mobile loop that straddles protease cleavage sites at the protein
surface(5, 13) . On the basis of the effect on the NMR
spectrum of cleavage of Th
by trypsin at
Lys
-Thr
and
Lys
-Glu
, the particularly clear resonances
at 6.82 and 7.12 ppm in wild-type Th
were assigned to 3,5
and 2,6 ring protons of Tyr
(5) . These resonances
were absent in both the Tyr
Asn and Tyr
Phe mutants (Fig. 1). Evidently, resonances from
other Tyr residues (at positions 120, 146, and 154) are very broad, as
expected for residues in a molecule having the correlation time of an
80-kDa protein like the Th
dimer. The appearance of
resonances in the Tyr
Asn mutant, between 2.6 and
2.8 ppm, corresponding to the
CH
of the Asn residue
introduced into the loop, was obscured by those from dithiothreitol
(and its breakdown products) in the sample. However, in the Tyr
Phe mutant, new narrow resonances were observed at 7.26
and 7.33 ppm, characteristic of ring protons of Phe. These resonances
were considerably sharper than those in wild-type protein at 7.33 ppm,
which were also attributed to Phe (possibly Phe
).
Figure 1:
One-dimensional NMR spectra of
wild-type Th and the Tyr
Asn and
Tyr
Phe mutants. A, NMR spectrum of 220
µM wild-type Th
(512 transients), in 10 mM [
H]Tris-Cl, p
H7.6, 10 mM (NH
)
SO
, 0.5 mM dithiothreitol in
H
O at 20 °C. B, 252 µM of the Tyr
Asn
mutant. C, 280 µM of the Tyr
Phe mutant.
Unexpectedly, the two Tyr substitutions both had
effects on resonances previously assigned to the CH
groups
of Met residues (Fig. 1). The NMR spectra of wild-type Th
at 20 °C reveal sharp resonances attributable to Met residues
at 1.97 and 2.04 ppm with shoulders at 2.06 and 2.08
ppm(5, 13) ; they are designated MetA, MetB, MetC, and
MetD, respectively (Table 1). In the Tyr
Asn
mutant, MetB was unaffected, but MetA was shifted downfield by 0.04
ppm, and MetC was shifted 0.02 ppm downfield to overlap with MetD at
2.08 ppm. In the Tyr
Phe mutant, MetB again was
unaffected, MetA was again shifted downfield (but only by 0.02 ppm),
and a slight downfield shift of MetC resulted in a peak at 2.065 ppm
with a shoulder at 2.08 ppm (MetD, Table 1). In the Tyr
Phe mutant, but not the Tyr
Asn
mutant, the region of the Ala resonances was resolved into two distinct
components (each possibly comprising one or more Ala
CH
doublets). Note that trypsin treatment of wild-type protein also
resulted in separation of two Ala components (5) ; the mobile
loop region includes several Ala residues.
Resonances other than
those assigned to Tyr and Met (and, in the Tyr
Phe
mutant, Ala) were indistinguishable in mutant and wild-type proteins.
Thus, the CH
of Thr at 1.25 ppm and ring protons of Phe at
7.37 ppm (although these were obscured in the Tyr
Phe mutant), the tentative Gly CH
at 3.96 ppm and Glu
CH
at 2.31 ppm, the resonances from unassigned amino
acid residues at 7.6-7.9 ppm, fine structure superimposed on the
broad methyl absorption at 0.8-1.0 ppm, and the ring-shifted
methyl protons at approximately 0.17, 0.30, and 0.63 ppm were all
essentially unchanged in both mutants.
Figure 2:
The effect of nucleotides on the NMR
spectrum of the Tyr
Phe mutant of Th
.
For conditions, see Fig. 1. A, 280 µM purified protein, no further additions; B, plus 30
µM NADH; C, plus 200 µM NADH; D, plus 200 µM NAD
.
In wild-type Th a
two-step binding reaction is revealed in NMR spectra recorded during
nucleotide titrations(5, 13) . It is characterized by
specific broadening of MetA at low concentrations of nucleotide,
followed, at higher concentrations, by broadening of other resonances
assigned to the mobile loop. In titrations with NADH the two-step
reaction is barely perceptible at 20 °C, although easily resolved
at 37 °C(13) . In the Tyr
Phe mutant
Th
(Fig. 3), a similar sequence of events was
observed to that in wild-type. Two differences were: (a) the
new Phe resonances at 7.26 and 7.33 ppm broadened during the titration
in the same way that the Tyr resonances broadened in wild-type protein,
and (b) of the two Ala resonances, split in the mutant, the
more upfield was more sensitive to broadening by NADH. Probably because
in the Tyr
Asn mutant the MetA resonance is
displaced downfield in the absence of nucleotides (see Table 1),
the two-step binding reaction was clearly observed in NADH titrations
even at 20 °C (Fig. 2). Thus, 30 µM NADH led to
more extensive broadening of MetA than, for example, the Ala or the Thr
resonances. Higher concentrations (200 µM) did lead to
broadening of the latter. The dependence of resonance broadening on
NADH concentration in both mutants was similar to that with wild-type
protein. NMR spectra recorded in titrations of mutant Th
with the analogue, AcPdADH, were qualitatively similar to those
from NADH titrations (not shown).
Figure 3:
The effect of nucleotides on the NMR
spectrum of the Tyr
Asn mutant of Th
.
For conditions, see Fig. 1. A, 292 µM purified protein, no further additions; B, plus 30
µM NADH; C, plus 200 µM NADH; D, plus 280 µM NAD
.
Also reflecting ligand-protein
interaction, linewidths of the NADH (and AcPdADH) resonances remained
broad during titration against both of the mutant proteins until added
nucleotide reached concentrations approaching 10M (i.e. in considerable excess of protein
concentration). Similar behavior was observed with wild-type protein
and was suggested to result from decreased mobility of NADH in its
protein-bound state and to an intermediate/fast exchange(5) .
In titrations of the mutant proteins with NAD, the
two-step reaction observed with wild-type protein (5) was again
evident. Thus, moderately low concentrations of oxidized nucleotide had
a specific effect on the MetA resonance, before other mobile loop
resonances were broadened ( Fig. 2and Fig. 3). In the
Tyr
Asn protein, in which the MetA resonance was
displaced downfield (see above), addition of NAD
led,
not only to broadening, but also to a shift back upfield that was more
extensive than that in wild-type Th
(Fig. 2; compare (5) ). Whereas the concentration dependence of resonance
broadening was similar in NADH titrations for wild-type Th
and for mutant proteins, this was not the case with
NAD
, where higher concentrations were required for
both mutants to give the response observed in the wild type.
In
marked contrast to the considerable broadening of NADH resonances in
the presence of either wild-type (5, 13) or mutant
Th (above), NAD
resonances became evident
in the wild-type titration spectra even at quite low concentrations,
consistent with a higher K
value for oxidized
nucleotide and faster exchange. In titrations with Tyr
Asn and Tyr
Phe mutants (Fig. 2D and 3D), NAD
proton
resonances were detectable at even lower concentrations of nucleotide,
providing another indication of its weaker binding.
As with
wild-type Th, NMR spectra of the mutants titrated with
AcPdAD
and with 5`-AMP were similar to those with
NAD
(not shown). Notably, they revealed a two-step
binding process; the MetA resonance was affected at lower
concentrations of nucleotide than the other loop resonances. As with
NAD
, higher concentrations of both AcPdAD
and 5`-AMP were required with both mutant proteins to produce an
equivalent broadening of the narrow resonances, and again, nucleotide
resonances were resolved at lower concentrations in the mutant than in
wild-type titrations.
As in wild-type protein, fluorescence from Trp in the
Tyr
Asn and Tyr
Phe mutants
was quenched upon addition of NADH. The dependences of fluorescence
quenching on nucleotide concentration were broadly similar to that in
wild-type (Fig. 4). Note, however, that because the K
is quite high, this analysis does not
sensitively detect decreases in binding affinity. In stopped flow
experiments (data not shown), it was observed that the time course of
Trp
fluorescence quenching by NADH was similar in the
Tyr
Asn mutant to that for wild-type
Th
(4) . Judging by the quenching of Trp
fluorescence, wild-type Th
bound AcPdADH with a lower
affinity than NADH(13) . Fig. 4shows that the
Tyr
Asn and Tyr
Phe Th
mutants also bound AcPdADH with a low affinity. Because of
limitations imposed by inner filtering by the nucleotide (see above),
it was difficult to estimate precise K
values. In
common with wild-type Th
, addition of either NAD
or NADPH led to no fluorescence quenching up to about 60
µM.
Figure 4:
The quenching of fluorescence of
Trp in mutant Th
by nucleotides. Experiments
were carried out in a medium containing 10 mM Tris-HCl, pH
8.0, 10 mM (NH
)
SO
, 1
mM dithiothreitol. The inner-filtering effect of the
nucleotides were corrected, as described.
, NADH;
, AcPdADH;
, NAD
. A, 0.6 µM Tyr
Asn mutant; B 0.6 µM Tyr
Phe mutant.
The
ability of wild-type and mutant Th to reconstitute reverse
transhydrogenation activity to R. rubrum membranes depleted of
native Th
is compared in Fig. 5. Depleted membranes
were prepared by salt washing chromatophores isolated from a strain of R. rubrum that overexpresses wild-type transhydrogenase (see
``Materials and Methods''). Experiments were performed with
close-to-saturating concentrations of nucleotide substrates. Rates of
reverse transhydrogenation with the Tyr
Asn and
Tyr
Phe mutants of Th
were about 18%
and 44%, respectively, of wild-type protein, but docking affinities
revealed by double-reciprocal plots (data not shown) were undiminished.
Figure 5:
Reconstitution of depleted membranes with
wild-type and mutant Th. Chromatophores from a strain of R. rubrum that overexpresses wild-type transhydrogenase were
washed with concentrated salt to remove their native Th
(see ``Materials and Methods''). The washed membranes
were resuspended (to a final concentration of 0.6 µM bacteriochlorophyll) in 100 mM Mops, pH 7.2, 50 mM KCl, 2 mM MgCl
, 1 µM carbonylcyanide-p-trifluoromethoxyphenyl hydrazone.
Th
was added to give the final concentration shown. The
reduction of AcPdAD
was measured at 375-450 nm.
, wild type Th
;
, the Tyr
Asn mutant;
, the Tyr
Phe
mutant.
Dependences of the rate of reverse transhydrogenation on the
concentration of AcPdAD (saturating NADPH) in the
reconstituted systems of depleted chromatophores, and either wild-type
or mutant Th
, are shown in Fig. 6A.
Double-reciprocal plots (not shown) yielded K
values for AcPdAD
of approximately 800, 600, and
60 µM in the Tyr
Asn and Tyr
Phe mutants and wild type, respectively.
Figure 6:
The
dependence on nucleotide concentration of the rate of AcPdAD reduction by NADPH by wild-type or mutant Th
reconstituted with depleted membranes. Conditions were as in Fig. 5, but using a Th
concentration of 50
nM, and (A) 200 µM NADPH with variable
AcPdAD
, and (B) 1.1 mM AcPdAD
with variable NADPH.
, wild type
Th
;
, the Tyr
Asn mutant;
, the Tyr
Phe
mutant.
Because the K values for AcPdAD
in the
mutants were high, it was not practicable to carry out experiments with
saturating concentrations of this nucleotide. Thus, Fig. 6B shows the dependence of reverse transhydrogenation rate on NADPH
concentration at 1.1 mM AcPdAD
.
Double-reciprocal plots (not shown) gave an approximate apparent K
for NADPH of 15 µM for the
Tyr
Asn mutant, 15 µM for the
Tyr
Phe mutant, and 30 µM for
wild-type Th
.
Rates of light-driven reduction of
thio-NADP by NADH (forward transhydrogenation) in
depleted chromatophores reconstituted, either with mutant or wild-type
Th
, were also investigated (Fig. 7). The light
drives photosynthetic electron transport, generating a proton
electrochemical gradient, which leads to enhanced proton flux through
transhydrogenase in its physiological (forward) direction. Depleted
membranes were prepared from wild-type chromatophores washed under mild
conditions to remove Th
whilst preserving coupling
activity. Because the overexpressing strain could not be used, the
level of accuracy was lower than in Fig. 6. Fig. 7shows
that maximum rates of light-driven forward transhydrogenation in
reconstituted systems were about 2-fold lower for both mutants than for
wild-type Th
. Dependences of rates of forward
transhydrogenation on nucleotide concentrations (Fig. 7, A and B) show that in both mutants the K
values for thio-NADP
and for NADH were not
significantly different from wild-type K
values of
approximately 5 and 4 µM, respectively.
Figure 7:
The
dependence on nucleotide concentration of the rate of
thio-NADP reduction by NADH during illumination by
wild-type or mutant Th
reconstituted with depleted
membranes. Chromatophores from a wild-type strain of R. rubrum were washed under mild conditions to remove their native Th
(see ``Materials and Methods''). Washed membranes were
resuspended (to a final concentration of 10 µM bacteriochlorophyll) in 100 mM Tris-HCl, pH 8.0, 2 mM MgCl
. Th
was added to give a final
concentration of 50 nM. The reduction of thio-NADP
was measured at 395-450 nm.
, wild type
Th
;
, the Tyr
Asn mutant;
, the Tyr
Phe mutant. A, 200
µM NADH with variable thio-NADP
; B, 40 µM thio- NADP
with
variable NADH.
Fig. 8shows that mutant Th proteins displaced the
wild-type protein from its binding site on chromatophores. Addition of
wild-type Th
to chromatophores led to a small increase in
the rate of reverse transhydrogenation, presumably because some domain
I protein was lost from the membranes during
preparation(1, 4) . However, addition of either the
Tyr
Asn or the Tyr
Phe mutant
Th
resulted in substantial loss of activity, indicating
that association-dissociation of domain I with domains II/III of
transhydrogenase can occur on the time scale of the experiment.
Figure 8:
Exchange of soluble and membrane-bound
Th. Chromatophores from a wild-type strain were resuspended
(to a final concentration of 10 µM bacteriochlorophyll) in
50 mM Mops, pH 7.2, 50 mM KCl. Purified Th
was added to give the concentration shown. The reduction of
AcPdAD
was measured at 375-450 nm.
,
wild type Th
;
, the Tyr
Asn
mutant;
, the Tyr
Phe
mutant.
By substituting Tyr of wild-type Th
with Phe and Asn, we have tested our prediction that
H NMR signals at 6.82 and 7.12 ppm in the wild-type protein
are attributable to that residue. Complete loss of those resonances
from the spectra of both mutants unambiguously confirms the assignment.
New, well defined resonances at 7.26 and 7.33 ppm in the Tyr
Phe mutant are characteristic of Phe ring protons, and
further indicate that the Phe has adopted the mobile nature of the
original Tyr. The emergence of new resonances in the Tyr
Asn mutant was masked by dithiothreitol present in the
sample.
Substitution of Tyr for Asn also led to
changes in resonances assigned to Met residues, notably a marked
downfield shift of MetA and a smaller shift in MetC. The assignment of
these residues is not yet possible, but is pertinent because the
behavior of the MetA resonance reflects events at an intermediate stage
of nucleotide binding ( (5) and (13) ; see below). It
was suggested (5) that MetA might derive from
Met
, and experiments are now in progress to test this.
Substitution of Tyr
for Phe also led to shifts of the
MetA and MetC resonances and to separation of Ala resonances. The fact
that the amino acid residue at position
influences the
NMR-detectable Met and Ala residues indicates that there is structural
organization in the loop even in the absence of nucleotides, but the
nature of the interactions is not understood. Evidently protons of the
Ala, MetA, and MetC residues can sample more than one environment on
the NMR time scale; the effect of the Tyr
substitution
might be to alter the exchange rate between different conformations, or
it might result in changes in the chemical shift of the Met and Ala
resonances in one of the conformational states, e.g. by
altering positions of charged or aromatic groups relative to methyl
groups of the amino acid residues.
The change from Tyr to Asn or to Phe in Th
is not accompanied by gross
changes in molecular structure; the protein retains its ability to form
dimers and to dock with the domain II/III components of
transhydrogenase, the short-wavelength emission of Trp
is
preserved, and, on the basis of NMR spectra, the protein fold and
environments of amino acids in the mobile loop (with the exception of
MetA, MetC, and Ala residues) are unaffected. Thus, effects of the
mutations on nucleotide binding and catalytic properties of the enzyme
are likely to be a direct consequence of altered properties of the
loop.
For both mutants, higher concentrations of NAD than for wild-type Th
were required to broaden
resonances ascribed to the mobile loop. This might mean either (a) that the K
for NAD
is increased by the amino acid substitution, or (b) that
differences in exchange rate(s) between Th
,
Th
-NAD
, and
Th
-NAD
(see(5) ) alter
linewidths without affecting the affinity for nucleotide. Similar
observations and interpretations apply also to AcPdAD
and 5`-AMP in wild-type and mutants. The very large K
for AcPdAD
of both the mutant
proteins, relative to the wild-type, during reverse transhydrogenation
(after reconstitution with Th
-depleted membranes) might be
another indication of increased K
for oxidized
nucleotide. Thus it is possible that Tyr
contributes to
the binding affinity of Th
for NAD
,
AcPdAD
, and 5`-AMP. This is consistent with the
observation that, in the two-dimensional
H NMR spectrum of
wild-type 5`-AMP-Th
complex, NOE interactions were detected
between Tyr
and bound nucleotide(13) .
It
cannot be determined with confidence whether or not the Tyr
Asn or Tyr
Phe mutations had a
significant effect on binding of reduced nucleotides by Th
.
There were no clear differences between the mutant and the wild-type
proteins in either the K
values for NADH in
forward transhydrogenation (Fig. 7) or the dependences on NADH
of the protein-NMR spectra ( Fig. 2and Fig. 3), but
neither of these give an unambiguous indication of the K
. The quenching of Trp
fluorescence
by reduced nucleotides (Fig. 4) must also be interpreted with
care; because of inner filtering, K
becomes more
subject to error as its value increases beyond 10-20
µM. However, it is reasonable to conclude that
substituting Tyr
with either Asn or Phe does not have a
large effect on the affinity of Th
for NADH or AcPdADH. The
fact that, in NADH titrations, the new Phe resonances in the
Tyr
Phe mutant broadened in a similar way to Tyr
resonances in wild-type Th
, indicates that the residue can
participate in mobile loop closure either with, or without, the 4-OH
group. There were minor differences in the NMR spectra recorded in NADH
titrations that arose from the fact that alteration of Tyr
caused shifts in Met and Ala resonances (see above), but
evidently the perturbations were not enough greatly to affect binding
affinity or loop closure.
Mutation of Tyr to either
Asn or Phe led to decreases in k
for both
forward and reverse transhydrogenation, when the Th
was
reconstituted with depleted membranes ( Fig. 6and Fig. 7). The loop clearly has a role in catalysis in addition to
its fine-tuning effect on the binding affinity of the protein for
nucleotide. Whether this is in the hydride transfer reaction, or in
conformational coupling with domain II/III components of
transhydrogenase, is not known. Although the Tyr
Phe mutant Th
had a substantially decreased activity, the
Tyr
Asn protein was considerably more inhibited,
particularly in reverse transhydrogenation. This might indicate that
both the aromatic ring and the 4-OH group of Tyr
are
important in the conformational dynamics of the loop in catalysis by
wild-type protein.
The equivalent of Tyr in bovine
transhydrogenase (Tyr
) is sensitive to modification by
5`-[p-(fluorosulfonyl)benzoyl]adenosine(22) .
Modified enzyme had reduced catalytic activity; NADH protected against
modification. On the basis of this, the equivalent residue in E.
coli transhydrogenase (Tyr
) was substituted with
His, Leu, Phe, and Asn(6) . In crude membrane fractions
isolated from bacteria carrying the mutation, specific activities
(mg
membrane protein) of AcPdAD
reduction by NADPH were 33% (or 51% in another strain), 38%, 45%
and 42%, respectively, lower than rates in membrane fractions prepared
from bacteria carrying wild-type transhydrogenase gene; K
values for AcPdAD
for the His,
Leu, and Phe mutant membranes were, respectively, 3-, 1.9-, and 3-fold
larger than those in wild-type membranes. Comparison between mutant and
wild-type transhydrogenase in that study is complicated by uncertainty
about the level of expression of the enzyme; the transhydrogenase
content of the bacterial membranes was assessed from their appearance
on SDS-polyacrylamide gels. Nevertheless, results of those experiments
are broadly consistent with results reported here. In our experiments
the statistical significance is assured because we used Th
purified to homogeneity, and the same preparation of
membranes for reconstitutions with both wild-type and mutant proteins.
Because the domain I protein of E. coli does not exist as a
discrete polypeptide, this strategy is unavailable in that system. In
our experiments, reverse transhydrogenation activities of Tyr
mutants of R. rubrum transhydrogenase were more
inhibited, relative to wild-type, than Tyr
mutants of E. coli transhydrogenase, and K
values of
the mutants were increased by a much larger factor. There might also be
real species differences between the two enzymes. The GYA motif of the
loop (5) is conserved in known transhydrogenase sequences, but
there is only low homology among other residues in the region; there
are no other invariant amino acid residues, although small and charged
residues preponderate. There is often greater variation in amino acid
sequence of surface loops because individual residues make only a small
contribution to the global structure. It is likely in transhydrogenase
that, during closure, multiple contacts are made between the loop and
the rest of the protein or bound nucleotide, and therefore, in
individual species, single amino acid substitutions in the loop might
have greater or lesser effects on catalysis. Uniquely in the R.
rubrum enzyme effects of mutations on nucleotide binding,
interaction between domains, conformational dynamics of the mobile
loop, and the Michaelis parameters can be assessed separately. The
present experiments establish that Tyr
is important in
the dynamics of mobile loop closure, and that substitution of the
residue profoundly affects the Michaelis parameters, thus, for the
first time, establishing a pivotal role for loop closure in catalysis.