However, the tight binding of NADPH, results in low reactions rates for the reverse and forward reactions catalyzed by the dI + dIII complex, as these reactions are respectively reviewed
Trang 1Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I–domain III interactions in proton-translocating
1
Department of Biochemistry, Go¨teborg University, Sweden;2Department of Molecular Biotechnology, Chalmers University of Technology, Go¨teborg, Sweden
Membrane-bound transhydrogenases are conformationally
driven proton-pumps which couple an inward proton
NADH (forward reaction) This reaction is stimulated by an
electrochemical proton gradient, Dp, presumably through an
increased release of NADPH The enzymes have three
domains: domain II spans the membrane, while domain I
and III are hydrophilic and contain the binding sites for
NAD(H) and NADP(H), respectively Separately expressed
domain I and III together catalyze a very slow forward
reaction due to tightly bound NADP(H) in domain III
With the aim of examining the mechanistic role(s) of loop
D and E in domain III and intact cysteine-free Escherichia
con-served residues bA398, bS404, bI406, bG408, bM409 and
bV411 in loop D, and residue bY431 in loop E were selected
In addition, the previously made mutants bD392C and
bT393C in loop D, and bG430C and bA432C in loop E, were included All loop D and E mutants, especially bI406C and bG430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of
indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhi-bition of both forward and reverse rates Most domain III mutants also showed a decreased affinity for domain I The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the inter-action between domain I and domain III
Keywords: transhydrogenase; NADP; proton pump; mem-brane protein
Transhydrogenase is a membrane protein, which is found in
the inner membrane of mitochondria and in the cytoplasmic
by NADH to the electrochemical proton gradient (Dp)
according to the reaction
ð1Þ
‘Out’ and ‘in’ denote the cytosol and matrix, respectively,
in mitochondria and periplasmic space and cytosol,
respect-ively, in bacteria Key features of this reaction is that?p
some 10-fold and causes a shift in the apparent equilibrium
constant from 1 to approximately 500 [1]
Transhydrogenase from Escherichia coli is composed of
an a subunit of about 54 kDa and a b subunit of about
other membrane-bound transhydrogenases, the E coli enzyme is composed of three domains Domain I (dI) and domain III (dIII) are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively, whereas domain II (dII) spans the membrane The genes encoding the hydrophilic and nucleotide-binding domains
of transhydrogenase from several species have been overexpressed and the proteins have been purified and characterized In all cases, dI is purified as a dimer and lack bound substrates Separately expressed dIII exists as a monomer and contains tightly bound NADP(H), reflecting
a dramatically increased affinity for NADP(H) as com-pared to the intact enzyme Even in the absence of the membrane bound dII, dI and dIII from the same or different species form a catalytically active complex capable of catalyzing the various transhydrogenation reactions However, the tight binding of NADP(H), results
in low reactions rates for the reverse and forward reactions catalyzed by the dI + dIII complex, as these reactions are
respectively (reviewed in [2–4])
The 3D structures of dI from Rhodospirillum rubrum [5] and dIII from bovine [6], human [7], E coli [8–10] and
by both X-ray crystallography [5–7] and NMR [8–12] The global fold of dIII is a six-stranded parallel b sheet
Correspondence to J Rydstro¨m, Department of Biochemistry,
Go¨teborg University, Box462, 405 30 Go¨teborg, Sweden.
Fax: + 46 31 7733910, Tel.: + 46 31 7733921,
E-mail: jan.rydstrom@bcbp.gu.se
Abbreviations: dI, transhydrogenase domain I; dIII, transhydrogenase
domain III; ecI, E coli dI; ecIII, E coli dIII; rrI, R rubrum dI;
rrIII, R rubrum dIII; cfTH, cysteine-free transhydrogenase;
AcPyAD+, oxidized 3-acetylpyridine-NAD+MIANS,
2-(4¢-malei-midylanilino)naphthalene-6-sulfonic acid (sodium salt).
(Received 30 May 2002, revised 12 July 2002,
accepted 26 July 2002)
Trang 2surrounded by helices and irregular loops All dIII
a nonclassical binding mode of the substrate, i.e as
3D structure of dIII with bound NADPH is still unknown,
even though NMR experiments in which NADPH was
added provided some information regarding regions that
were affected by a change in the redox-state of the substrate
[9–11] The crystal structure of dI from the R rubrum
transhydrogenase [5] showed that it was a dimer, with the
monomer comprised of the two subdomains 1 A and 1B [5]
The 1B subdomain has a Rossman fold responsible for
binding NAD(H) in a novel mode as compared to other
NAD(H)-binding enzymes [5]
By using NMR in combination with mutagenesis, an
extensive characterization of the dynamic interface between
recently carried out [10] In addition to information
regarding important residues involved in the ecI–ecIII
interface, the results revealed unexpected redox-dependent
changes of the ecI–ecIII interface suggested to be relevant
for the overall reaction mechanism of the intact enzyme
The regions at the C-terminal end of the b sheet comprised
of residues bG389-bI406 (part of loop D, linking b-strands 4
and 5) and bG430–bV434 (part of loop E, linking b strands
5 and 6) were identified as redox-sensitive regions, regulated
by the redox-state of NADP(H), that strongly influenced
complex from R rubrum has recently been resolved by
X-ray crystallography [13], which revealed dI–dIII
inter-faces at an atomic level similar to those derived from the
NMR studies [10] Based on the crystal structures of
com-plex from R rubrum [13], loops D and E have previously
been suggested to be important in NADP(H)-binding
and possibly also coupling to proton translocation
[4,13] The corresponding regions in ecIII are indicated in
an NMR-derived structural model (Fig 1) and in the
amino-acid sequence with secondary structure elements
(Fig 2)
The functional importance of loops D and E have so far
not been functionally established In the present work, the
roles of these regions were investigated in greater detail
using site-directed mutagenesis The results suggest that
loop D is important in communicating affinity changes in
the NADP(H)-binding site to domain I, and that both loop
D and E regulate the release of NADP(H) In support of the previous suggestion [4,13] both loops are suggested to play a key role in the regulation of the enzyme by an electrochem-ical proton gradient
M A T E R I A L S A N D M E T H O D S
Site-directed mutagenesis The Quikchange mutagenesis kit (Stratagene) was used to introduce single cysteine mutations in isolated ecIII and cfTH A modified pET8c plasmid used for the prepar-ation of histidine-tagged ecIII [14] served as a DNA template in the construction of the seven cysteine mutants ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIY431C In addition, four previously produced mutants, i.e ecIIID392C [15], and ecIIIT393C, ecIIIG430C and ecIIIA432C [9], were further characterized The cfbG430C mutant was based
on the pCLNH plasmid to which an N-terminal histi-dine tag has been added to the cfTH gene [14] The correctness of the mutant products was checked by DNA sequencing
Fig 1 Two views of the partial 3-dimensional structure of ecIII The NADP(H)-binding site
of ecIII This illustration was based on the NMR structure of E coli dIII [10] The beginning and end of loop D (excluding a5) and E are P403-V411 and M427-G433, respectively The illustration was prepared using the software [26].
Fig 2 The amino-acid sequence of ecIII and secondary structure ele-ments based on NMR data Secondary eleele-ments were based on results from NMR experiments [10] Black shaded residues are conserved, and green-shaded residues are similar among different species of transhy-drogenase.
Trang 3Expression and purification
The gene encoding ecIII was based on the 177 C-terminal
residues of E coli transhydrogenase b-subunit
(bQ286-bL462) EcIII mutants [14] and E coli cysteine-free
tran-shydrogenase enzymes [15] were expressed and purified as
described The plasmid pCD1 encoding R rubrum domain I
(rrI) was expressed in E coli TG1 cells [16] and purified
according to the method described by Bizouarn et al [17]
with modifications After sonication and centrifugation of
1 L culture, the supernatant was loaded onto a 15-mL
Q-Sepharose HP column (Pharmacia) equilibrated with
was eluted with about 60 mL of the same buffer, after which
After 10 h of incubation the sample was centrifuged for 1 h
at 18 000 r.p.m in a Beckman JA20 rotor, and the
supernatant loaded onto a 15-mL Butyl Toyopearl column
(Tosohas) Protein was eluted with a gradient (300 mL) of
All domains displayed a purity greater than 90% as
judged by SDS-polyacrylamide gel electrophoresis using
8–25% gradient gels in the Phast system (Pharmacia) for
transhydrogenase domains and 10–20% gradient gels
(Novex) for the cfTH enzymes
Determination of protein concentration and substrate
content
Protein concentrations were determined using the
bicinch-oninic acid assay with bovine serum albumin as standard
[18] The content of bound NADPH in the ecIII mutants
was determined by absorbance spectroscopy at 339 nm,
Klingenberg procedure as described previously [14]
Activity assays
Unless stated otherwise, transhydrogenation reactions
cata-lyzed by mutant and wild-type ecIII were assayed as
titrations were performed in which the ecIII concentration
was kept constant and the rrI concentration varied until a
maximal rate was reached
The forward and reverse reactions catalyzed by the cfTH
and cfTH mutant enzymes were measured as described [20]
and cyclic reactions were followed optically at 375 nm as
reaction was measured spectroscopically at 398 nm as
For comparative reasons, the transhydrogenation
reac-tions catalyzed by rrI + wild-type and mutant ecIII
mixtures were also assayed in the cfTH assay buffers All
Fluorescence measurements Fluorescence measurements were carried out on a SPEX Model FL1T1 s2 and Shimadzu RF5001PC
section was used and the excitation and emission slits were both 2.5 nm
Determination of the NADPH release rate from ecIII
by fluorescence The release rate of NADPH from ecIII was determined from the exponential decrease in fluorescence as bound NADPH was released from ecIII and oxidized by glutathi-one and glutathiglutathi-one reductase using excitation and emission wavelengths of 340 and 460 nm, respectively Oxidized
reductase; the fluorescence was monitored for up to 20 min
In the case of the ecIIIV431C mutant, it was preincubated
for 5 min prior to the assay The measurements were carried
(pH 7.0)
R E S U L T S
Characterization of single-cysteine mutations in ecIII
In order to elucidate the mechanistic roles of loop D and
E in greater detail, the single ecIII cysteine mutants,
ecIIIM409C, ecIIIV411C (loop D), and ecIIIY431C (loop E), were expressed in the cysteine-free background and purified as described in Materials and methods In addition, the previously made mutants ecIIID392C and ecIIIT393C
in loop D, and ecIIIG430C and ecIIIA432C in loop E, were included All mutants were characterized with respect to substrate binding, catalytic activities of the different trans-hydrogenation reactions and affinity for rrI In these assays and under the conditions used dimer formation by all mutants amounted to less than 10% as tested by SDS/ PAGE (data not shown)
Figure 3 A and B show the detailed positions of some of the mutated as well as other important residues in ecIII, and
Note that loops D and E are much more defined in the crystal structure of bovine dIII shown in Fig 3 than in Fig 1 A recently produced high resolution NMR structure
Johansson, A Pedersen, J Rydstro¨m and B G Karlsson, unpublished results)
Content of bound NADP(H) in dIII The increased affinity of dIII for NADP(H) is reflected in the content of tightly bound NADP(H) in almost 100% of the molecules of separately expressed ecIII [10,19] The presence of NADP(H) changes the absorbance maximum,
indication of the fraction of apo-protein An additional
Trang 4typical property of isolated ecIII is the percentage of bound
NADP(H), which is highly reproducible for different
preparations In order to assess the effects on
and NADPH were determined for the mutants generated in this investigation i.e ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIY431C (Table 1)
Fig 3 NADP(H) binding region of dIII viewed perpendicular to (A) and parallell with the
b sheet (B) The structure was modelled using
MOLMOL [26] based on the crystal structure of the bovine dIII with bound NADP + (PDB entry 1D40) Important residues and loops D and E, are indicated Numbering of mutated residues is according to ecIII.
Table 1 Content of bound NADP(H) in wild-type and mutant ecIII The concentrations of NADPH in the ecIII enzymes were calculated from UV-Vis spectra and the content of NADP+was determined by a modified Klingenberg procedure (see Materials and methods) The k max value corresponds to the wavelength at which maximal absorption was observed for the interval 200–400 nm in UV-Vis spectra.
Enzyme Loop affected k max NADP + (%) NADPH (%) Apo-form (%)
Trang 5All ecIII mutants contained bound substrate, where
with an increased content of NADPH This effect was
most pronounced in the case of the ecIIIS404C,
ecIIIM409C and ecIIIY431C mutants, with 33%, 27%
and 28% bound NADPH, respectively, and a relatively
ecIIIY431C did not follow the same pattern, but
contained approximately 54% and 29% apoprotein,
respectively (Table 1) Thus, it is clear that the mutations
in the redox-sensitive loops D and E strongly affected the
binding site
In all mutants in loop D, except ecIIIV411C, the
nucleotide content was at least 89%, with a 3–5 fold
increase in the percentage of bound NADPH (Table 1)
Clearly, the small fraction of apo-protein in these mutants
reflected the fact that this region of the protein is not directly
involved in substrate binding
Forward reaction catalyzed by rrI + ecIII mutant mixtures
NADH) catalyzed by rrI and ecIII mutant mixtures was
examined by protein-protein titrations in which the ecIII
concentration was kept constant and the rrI concentration
varied until a maximal rate was reached In this and other
dI-dependent assays in this investigation, rrI rather than ecI
was used due to the generally higher activities obtained with
this dI preparation (cf Fig 2) The [rrI]/[ecIII] ratio at
half-saturation is a measure of the affinity of the complex formed
for rrI and ecIII [10] and may be used to gain information
about the role of a particular amino-acid residue in the
interactions with dI However, because the rrI concentration
for the rrI + ecIII complex and the release rate of
thio-NADPH from domain III, it can not be considered as a true
measure of the affinity between the two domains Due to the
tight binding of nucleotides to wild-type ecIII, the forward
reaction catalyzed by ecIII with saturating concentrations of
rrI is limited at pH 7.0 by the slow release of thio-NADPH
from domain III [16] An increase in the forward reaction
rate may thus be regarded as an increase in the release rate
of NADPH
Table 2 summarizes the results obtained for
measure-ments of the forward reaction catalyzed by rrI and ecIII
mutants The maximal rates for the ecIIIG430C and
ecIIIY431C mutants were 275 and 100 times that of the
wild-type ecIII, respectively This pronounced increase
demonstrates the dramatic effect on the rate of dissociation
of NADPH caused by mutations in loop E Depending on
the position in which the cysteine residue was introduced,
mutations in loop D had different consequences on the
forward reaction rate The ecIIII406C mutant showed a
35-fold higher rate than wild-type ecIII, whereas the
ecIIIS404C mutation had a minor effect (Table 2)
Likewise, the ecIIIA398C, ecIIIG408C, ecIIIM409C,
ecIIIV411C and ecIIIA432 mutants showed relatively minor
changes As the ecIIII406 residue does not participate
directly in substrate binding [6,7], the increased forward
reaction rate demonstrated by the ecIIII406C mutant was
possibly a result of perturbation of the surroundings of this
position caused by the mutation An obvious candidate
responsible for this effect is the conserved bD392 residue,
-complexed dIII crystal structure [6,7]
The [rrI]/[ecIII] at half-saturation was increased for all cysteine mutants, but appeared to be correlated to the maximal rate displayed by the mutants (Table 2)
Reverse reaction catalyzed by rrI + ecIII mutant mixtures Analyses of the reverse reaction catalyzed by rrI + ecIII mutant mixtures were performed by protein-protein titra-tions in the same way as for the forward reaction Like the forward reaction, the reverse reaction is limited at pH 7.0 by the slow release of the product bound to dIII [16], but is several-fold faster The maximal rate of the reverse reaction
is thus an excellent tool for examining if a mutation has
constant for the rrI + ecIII complex but, like the forward reaction, this ratio is also dependent on how fast the product
is released from ecIII An elevated release rate needs more dI
to saturate the reaction
As shown in Table 3 the maximal rates obtained for both the ecIIII406C and ecIIIY431C mutants were 4.5-fold higher than that of wild-type ecIII, indicating an increased
ecIIII406C mutant was probably an indirect effect caused
by perturbations in its environment The ecIIIS404C, ecIIIG408C, ecIIIM409C and ecIIIV411C mutations did not affect the reverse reaction rate significantly, which is consistent with the fact that these positions are not in the substrate binding-site [6,7] The [rrI]/[ecIII] ratios at half-saturation of the reverse reaction correlated well with the maximal rate of the ecIII mutants
Cyclic reaction catalyzed by rrI + ecIII mutant mixtures
NADH via NADP(H) bound to dIII, was analyzed by protein-protein titrations in which the ecIII concentration
Table 2 Properties of the forward reaction catalyzed by wild-type and mutant ecIII in the presence of rrI The values are estimations from protein-protein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown) The assays were performed as described in Materials and methods The [ecIII] values refer to the fixed enzyme concentration used in the titrations The [rrI] values correspond to the concentration of rrI at half V max
Enzyme
[ecIII]
(n M )
[rrI]
(n M )
[rrI]/
[ecIII]
V max
(mol thio-NADPH)Æ (mol ecIII))1Æmin)1 % ecIII 5000 5 0.001 0.04 100 ecIIIA398C 2500 3 0.001 0.12 300 ecIIIS404C 5000 4 0.0008 0.08 200 ecIIII406C 2500 20 0.08 1.4 3500 ecIIIG408C 2500 5 0.002 0.13 325 ecIIIM409C 5000 15 0.003 0.13 325 ecIIIV411C 5000 10 0.002 0.22 550 ecIIIG430C 2500 400 0.16 11 27500 ecIIIY431C 2500 140 0.06 4 10000 ecIIIA432C 2500 10 0.004 0.13 325
Trang 6was kept constant and the rrI concentration varied until the
reaction rate reached a maximum NADP(H) remains
bound to dIII during the entire catalytic cycle and the rate of
the cyclic reaction at pH 7.0 has been shown to be limited
by the hydride transfer steps [16] Measurements of the
cyclic reaction thus offers an opportunity to examine the
affinity of ecIII mutants for dI The [rrI][/ecIII] ratio at
half-saturation is only dependent on the dissociation constant for
the rrI + ecIII complex Using the actual rrI and ecIII
In Table 4 the data from the protein-protein titrations for
the ecIII mutants are listed Except for the ecIIIS404C
considerably higher in all of the mutants made in the
redox-sensitive loops The most affected mutants were
the ecIIIA398C (loop D), ecIIIM409C (loop D) and
ecIIIY431C (loop E) mutants which exhibited 6–15 times
wild-type ecIII (Table 4) In agreement with the crystal structure
loops D and E directly or indirectly make crucial contacts with dI Mutations in the I406–V411 region affected the hydride transfer efficiency and only 24–31% of the maximal rate of the cyclic reaction could be reached, despite saturating concentrations of rrI Interestingly, the ecIIIS404C and ecIIIA398C mutants were still able to catalyze the hydride transfer at a wild-type rate, even though the affinity for rrI had been substantially lowered
Release rate of NADPH measured by fluorescence
460 nm when using excitation at 340 nm, was utilized in order to determine the rate of release of NADPH from ecIII By this method bound NADPH was oxidized by glutathione reductase and glutathione The reaction is limited by the rate of release of NADPH and the resulting decrease in fluorescence could consequently be used to
glutathione and glutathione reductase of NADPH bound to ecIIIG432C and ecIIIG430C The rate obtained with ecIIIA432C is representative of the wild-type rate In contrast, the ecIIIG430C mutant showed a dramatic 110-fold increase in oxidation rate Based on similar oxidation
calculated (Table 5) The release rate of NADPH was only significantly increased for the ecIIII406C, ecIIIG430C and ecIIIY431C mutants In contrast to other mutants, the NADP(H) bound to ecIIIY431C was rapidly lost upon storage The NADPH released was subsequently oxidized, requiring reloading with equimolar NADPH prior to assay The ecIIII406C and ecIIIY431C mutants displayed a fourfold to fivefold faster release of NADPH These results also indicate that there is no obvious relationship between the release rate of NADPH and the content of bound NADP(H) (cf Table 1)
Characterization of the G430C mutant in intact
In order to examine the effects of a mutation in the redox-sensitive region of loop E in the intact E coli transhydro-genase, the cfTHG430C mutant was constructed and the
Table 3 Properties of the reverse reaction catalyzed by wild-type and
mutant ecIII in the presence of rrI The values are estimations from
protein-protein titration curves in which the ecIII concentration was
fixed and the rrI concentration varied (not shown) The assays were
performed as described in Materials and Methods The [ecIII] values
refer to the fixed enzyme concentration used in the titrations The [rrI]
values correspond to the concentration of rrI at half V max
Enzyme
[ecIII]
(n M )
[rrI]
(n M )
[rrI]/
[ecIII]
V max
(mol AcPyADH)Æ (mol ecIII))1Æmin)1 % ecIII 4900 20 0.004 4 100
ecIIIA398C 2500 30 0.012 4 100
ecIIIS404C 4000 30 0.008 3 75
ecIIII406C 2500 250 0.100 18 450
ecIIIG408C 2500 40 0.016 4 100
ecIIIM409C 5000 150 0.030 3 75
ecIIIV411C 2500 110 0.044 5 125
ecIIIY431C 2500 240 0.096 18 450
Table 4 Properties of the cyclic reaction catalyzed by wild-type and mutant ecIII in the presence of rrI The values are estimations from protein-protein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown) The assays were performed as described in Materials and Methods The [ecIII] values refer to the fixed enzyme concentration used in the titrations The [rrI] values correspond to the concentration of rrI at 1 / 2 V max K d is the estimated dissociation constant derived according to K d ¼ [rrI]-[ecIII]/2.
Enzyme
[ecIII]
(n M )
[rrI]
(n M )
K d
(n M )
V max
(mol AcPyADH)Æ (mol ecIII))1Æmin)1 %
Trang 7resulting mutant protein was characterized with respect to
catalytic activities The kinetic properties of the various
transhydrogenation activities catalyzed by the cfTHG430C
mutant are summarized in Table 6 The severe effect of
mutating this conserved glycine into a cysteine was clearly
reflected in the resulting maximal rates of the reverse,
forward and cyclic reactions which were all between 7 and
for NADPH in the reverse reaction was 40 times higher
unchanged Consequently, the cfTHG430C mutation
resul-ted in a substantial loss of affinity for NADPH, while the
For comparative reasons, the transhydrogenation
reac-tions catalyzed by rrI + wild-type ecIII and ecIIIG430C
enzymes mixtures and by cfTH and cfTHG430C enzymes,
Fig 4 Release of NADPH from ecIII mutants studied by fluorescence.
K offNADPH for ecIII mutants were estimated from curves obtained
when monitoring the decrease in fluorescence intensity as ecIII
enzymes were treated with glutathione and glutathione reductase (see
Materials and methods) Upper trace denotes ecIIIA432C and lower
trace ecIIIG430C.
Table 5 Release rates of NADPH from wild-type and mutant ecIII
determined by fluorescence The release rates of NADPH from ecIII
enzymes were determined by fluorescence as described in Materials
and Methods The K offNADPH was derived from curves obtained by
monitoring the decrease in fluorescence as NADPH was oxidized.
Enzyme
K offNADPH
(s)1)
Relative rate
ecIIIT393C 0.010 2
ecIIIS404C 0.002 0.4
ecIIII406C 0.020 4
ecIIIM409C 0.004 0.8
ecIIIG430C 0.560 110
ecIIIY431C 0.025 5
ecIIIA432C 0.011 2
Vma
+ ,2
+ ,
Vmax
Vmax
Vmax
1 Æmin
1 Æmin
m (lM
1 Æmin
Trang 8were all analyzed in buffer C The maximal rates listed in
Table 7 were obtained from the pH optima of the
respective mixtures of domains or enzymes For the
reconstituted system, rrI + ecIII, there was a pronounced
difference between the rates of the forward and reverse
reaction, the reverse rate being 150 times higher For the
reconstituted rrI + ecIIIG430C mutant, this difference in
rates had largely disappeared, the reverse reaction being
only 4.5 times faster (Table 7) Thus, the ratio between the
forward and the reverse reaction rates was approximately
the same for wild-type cfTH and cfTHG430C enzymes,
but the activities displayed by the cfTHG430C mutant
were only 8–10% of those catalyzed by wild-type cfTH
(Table 7) However, the maximal rates of the forward and
reverse reactions exhibited by the rrI + ecIIIG430C
complex and the cfTHG430C enzyme were almost the
same (Table 7)
D I S C U S S I O N
Conformational changes involved in the proton pumping
mechanism of transhydrogenase have been suggested to be
dependent on binding/release of NADP(H) and the
and the bonds stabilizing its association with dIII have been
complex [13]; the two forms of dIII do not reveal any major
Structur-ally, loop E is involved in the binding of the pyrophosphate
group as well as the ribose of 2¢-5¢-ADP through the
conserved bG430 The preceding conserved
K424-R425-S426 residues bind the 2¢-phosphate [6,7,13] The structural
role of loop D is less obvious, but it appears to interact with
loop E as well as dI [13], and the semiconserved I406 points
into an apparent crevice formed by loop D and E towards
I406 and the nicotinamide ring is about 6 A˚ [13] Based on
this structural information, it was proposed that loop D and
E ( the lid) were involved in the interaction with dI and
the occluded state to the open state and vice versa [2,4,13]
These conclusions were supported by NMR studies where
dIII [11] Studies of ecIII by NMR [10], especially chemical
shifts caused by the presence of ecI and/or NADP(H),
showed that the ecIII itself and the ecI–ecIII interface were
altered upon a change in the redox-state of the bound
NADP(H)
Despite the large amount of structural information
available for loops D and E, their functional roles have
not been systematically examined by site-directed
mutage-genesis Residues subjected to mutagenesis were therefore
chosen based on the magnitude of their chemical shift
perturbations in the NMR experiments [10], i.e especially
G389-I406 and G430-V434, and surrounding residues, and
on their conservation among 62 transhydrogenase gene
sequences Some of these mutants, i.e ecIIIK424C,
ecIIIH345C, ecIIIA348C, ecIIIR350C [15], and ecIIIT393C,
ecIIIR425C, ecIIIG430C and ecIIIA432C [19], were partly
characterized previously In order to be able to react these
with thiol-specific reagents, all selected residues were
mutated to cysteines in the cysteine-free E coli
1 Æmin
1 Æmin
1 Æmin
Trang 9The role of loop D
Mutations were introduced in loop D in ecIII, a region that
was suggested by NMR experiments to be involved in
redox-regulation of the interactions of ecIII with ecI [10] In
addition to ecIIIA398C, ecIIIS404C and ecIIII406C,
muta-tions were also made in the adjacent G408-M409-P410-V/
I411 region, i.e ecIIIG408C, ecIIIM409C and ecIIIV411C
Earler made mutants in this region include ecIIID392C [15]
and ecIIIT393 [10] All of these mutants show a varying
content of bound NADP(H), the most conspicuous being
ecIIIR425C [10] and ecIIID392C [15] which are isolated as
100% apo-form, and ecIIIV411C which has 54% apo-form
(Table 1)
Introduction of a cysteine in the S404 position of ecIII
had little or no effect on the substrate-binding properties
and affinity for dI This ecIII mutant behaved wild-type like
in all experiments It should be noted, however, that the
replacement of serine with cysteine is a rather mild
substitution When the ecIIIS404C mutant was reacted
with MIANS and NEM (A Pedersen, C Johansson and
J Rydstro¨m, unpublished results), the reverse reaction was
stimulated by a factor of 1.6 and 2.0, respectively, indicating
that the side-chain of S404 is pointing towards the substrate
The ecIIII406C mutation led to higher release rates for
reverse and forward reaction rates, as well as a faster release
of NADPH in fluorescence measurements These
observa-tions might seem peculiar as the I406 residue does not make
any specific interactions with NADP(H) in the crystal
structure [6,7,13] However, as a working hypothesis, it is
possible that I406 creates a suitable environment for the
essential substrate-binding D392 This aspartic acid residue
is located within 6 A˚ from I406 in the crystal structure and
has been proposed on the basis of mutagenesis to be a key
residue in catalysis/binding as well as the proton pumping,
possibly constituting one end of the proton wire
[2,10,15,20,23–25,27] Structural evidence [2,4,6,7] supports
these proposals I406 may take part in the regulation of the
accessibility of the D392 side-chain and thereby control
protonation events The rrI affinity was also affected by the
ecIIII406C mutation, as shown by the five-fold increase in
The local environment of I406C was recently
demonstra-ted by MIANS labeling experiments to depend on the
redox-state of the added substrate (A Pedersen, C
Johansson, B.G Karlsson & J Rydstro¨m, unpublished,
results) This difference might reflect a movement of the
I406C side-chain, and probably the entire loop D, that is
coupled to events in the NADP(H)-binding site
Mutations in the G408-M409-P410-V/I411 region did
not influence the substrate-binding characteristics of ecIII,
but caused a substantial decrease in the affinity for domain
higher than that for wild-type ecIII and the maximal rate of
the cyclic reaction was only about 24%, indicating that the
complex was distorted by this mutation
The role of loop E
Mutations in loop E in isolated ecIII had dramatic
consequences on its interactions with both dI and
NADP(H) The most remarkable property of these mutants
was the high dissociation rates of NADP(H), suggested by both high forward (Table 2) and reverse (Table 3) reaction rates, particularly the fast release of NADPH in fluores-cence measurements The ecIIIG430C and ecIIIA432C mutants were earlier shown to be catalyze 850- and 150-fold increased reverse rates, respectively [10] In the presence of rrI, the forward reaction catalyzed by the ecIIIG430C and ecIIIY431C mutants was 275 and 100 times faster, respect-ively, than that of wild-type ecIII As this reaction catalyzed
by the rrI + ecIII complex normally is limited by the slow release of the NADPH, these high rates indicate high dissociation rates of NADPH Indeed, measured directly, a
ecIIIG430C mutant
A careful analysis of the structure of ecIII revealed that a plausible explanation for the above observation is that the side-chains of G430-Y431-A432 appear to be involved in specific interactions with NADP(H) and thus could con-tribute to an increased affinity for this substrate (see [6,7,13]) The side-chain of a cysteine residue most likely adopts a different angle than those of the original GYA residues This is particularly apparent from the content of bound NADP(H) and percentage apo-form of the ecIII mutants In contrast to wild-type ecIII, both ecIIIG430C [10] and ecIIIA432C [10] have a reversed content of NADP(H), i.e predominantly NADPH with no or little
and all three mutants have high apo-form content
In addition to being important for the regulation of NADPH release, loop E also plays a crucial role in the interactions with dI This was demonstrated by the high concentration of rrI needed for half-saturation of the cyclic reaction catalyzed by the ecIIIY431C mutant (Table 4), as well as the ecIIIG430C and ecIIIA432C
rrI + ecIIIG430C complex was about 10 and 15 times, respectively, higher than that for the rrI + ecIII complex
As expected, the rate of the cyclic reaction was inversely
the rate of the reverse reaction catalyzed by the ecIIIG430C [10], ecIII/431C (Table 3) and ecIIIA432C [10] mutants was proportionate to the [rrI]/[ecIII] ratio at
The inherent tight binding of NADP(H) in isolated domain III has previously been explained by the hypo-thesis that separately expressed dIII mimics the ‘occluded’ conformation of domain III in the intact enzyme [2,4,16] This occluded conformation is assumed to correspond to a state in which the hydride transfer step takes place [2] Consequently, the activities of the reverse and forward reactions catalyzed by rrI + ecIII, in which release of NADP(H) is limiting, were very low as compared to that
of intact cfTH (Table 7) The comparison of the rates of the various transhydrogenation reactions catalyzed by wild-type and mutants of rrI + ecIII complexes and intact transhydrogenases allowed an important conclusion regarding the differences between isolated ecIII and dIII
as it functions in intact transhydrogenase The ratio of the rates of the reverse and forward reactions catalyzed by rrI + ecIIIG430C was similar to that for cfTHG430C, i.e approximately 7 (Table 6) Normally, this ratio is the same for cfTH, but 150 for rrI + ecIII (Table 6) Introduction of a cysteine residue in the G430 position
Trang 10of ecIII obviously perturbed the conformation of loop E
in such a way that allowed it to function essentially as in
the wild-type intact enzyme/cfTH, i.e with a strongly
increased rate of the forward reaction and a less strong
increase in the reverse reaction Indeed, this change is
higher dissociation rate of this substrate, assuming that the
evidence is rather indirect, it is conceivable that the tight
binding of NADP(H) in the occluded state of domain III
directly or indirectly involves G430 and/or the
conforma-tion of loop E An interesting possibility is therefore that
G430 and loop E in the resting state of the intact enzyme
(and in the ecIIIG430C mutant) are much less associated
with NADP(H), i.e the lid is open
As proton translocation in transhydrogenase is very likely
associated with conformational changes that affect binding
and release of NADP(H) [2,4], loop E may play a major role
in the coupling mechanism of transhydrogenase In
addi-tion, the changes in affinity for NADP(H) could be
communicated to domain I as loop E forms part of the
region that confers a redox regulation of the ecI + ecIII
complex interface
In conclusion, the present results suggest that loop D is
involved in the interactions with domain I and that the I406
residue is a potential candidate for the regulation of the
accessibility of the side-chain of the D392 residue that is
essential for proton-pumping Moreover, the results support
the notion that loop E functions as a mobile lid [4,7,13],
regulating the release of NADPH, a step that probably is of
central importance in the coupling mechanism of
transhy-drogenase It is proposed that movements of these two loops
work in concert to regulate the affinity of NADP(H),
protonation events in their surroundings and to
communi-cate these changes to domain I
A C K N O W L E D G E M E N T S
This work was supported by the Swedish Natural Science Research
Council AP acknowledges a grant from the Sven and Lilly Lawski
Foundation.
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