From the
Aldose reductase (ALR2) shows a strong specificity for its
nucleotide coenzyme, binding NADPH much more tightly than NADH (K
Aldose reductase (ALR2),
Kinetic studies have shown
that the enzyme follows an ordered sequential mechanism with the
coenzyme NADPH binding to the enzyme first(9, 10) .
Reduction of the aldehyde occurs with Tyr-48 donating a proton to the
carbonyl of the substrate (11, 12) and His-110 directing
the stereochemistry of the hydride transfer(13) . During the
reaction the enzyme
Our current studies focus
specifically on the binding interactions of the 2`-phosphate of NADPH
to ALR2. ALR2 has a strong specificity for its nucleotide substrate,
binding NADPH much more tightly than NADH (4). Binding interactions
between ALR2 and NADPH that are responsible for this specificity must
reside with the 2`-phosphate which is absent in NADH. The crystal
structures of recombinant hALR2(14) , as well as the hALR2-C298S
mutant(15) , both complexed with NADPH, show that the adenosine
mononucleotide is firmly secured by the interactions with its
2`-phosphate group. The phosphate forms hydrogen bonds with the
hydroxyl groups of Ser-263, Thr-265, and the main chain NH of Val-264
(14). It is also involved in salt links and hydrogen bonds with the
side chains of Lys-262 and Arg-268 residues that are also conserved
among other members of the aldo-keto reductase
family(14, 15) . Chemical
modification(22, 23) , together with x-ray
crystallographic studies, also implicate Lys-262 and Arg-268
interacting with the 2`-phosphate. Site-directed mutagenesis of Lys-262
to methionine in human ALR2 results in a large increase in K
In
this study, we investigate the contribution of Arg-268 to NADPH binding
and the transition state by comparing the kinetic and thermodynamic
properties of coenzyme binding (NADP(H) and NAD(H)) to the wild type
ALR2 and the R268M mutant.
Site-directed
mutagenesis of Arg-268 was performed using the Mutagene M13 In
Vitro Mutagenesis Kit (Bio-Rad) by following the manufacturers
protocol. The oligonucleotide used to generate the R268M mutation was
5`-CAGCAATCATTTCTGGTG-3`, and was hybridized to a single stranded M13
mp19/hALR2 cDNA insert containing a R268K mutation. The oligonucleotide
used to generate the R268K mutant was
5`-GTTCTCAGCAATCTTTTCTGGTGTCAC-3`. Both the WT and R268M-hALR2 cDNA
were completely sequenced after subcloning into the expression vector
to verify the desired mutation and to ensure that no other mutations
had occurred.
All
subsequent purification steps were carried out at 4 °C, as
described for native, muscle hALR2(31) . The purified enzyme was
dialyzed against 5 mM sodium phosphate, pH 7.0, containing 1
mM dithiothreitol and stored at 4 °C.
For kinetic studies, enzyme
activity was determined in 0.1 M sodium phosphate buffer, pH
7.0, containing the appropriate concentrations of substrates. The
reactions were started by addition of aldehyde, as suggested
previously(9) . The initial velocity was computed using a
quadratic fit to the progress curve from 0 to 120 s, to ensure
measurement of the initial steady-state rate(9) . The kinetic
constants for DL-glyceraldehyde reduction by WT in the
presence of NADH were determined from assays at 366 nm using a 5-mm
path-length cuvette, as were the constants determined for R268M-hALR2
in the presence of NADPH. For the reaction catalyzed by R268M in the
presence of NADH, the rates were determined at 386 nm. All standard
errors of fits were less than 30%.
The steady-state kinetic
constants and their standard errors were calculated by fitting the
equations in the hyperbolic form to the data with a least-squares
analysis using the Marquardt algorithm with the program Kinetics
Software (Hewlett-Packard HP89512).
For the WT, the kinetic
constants were determined by fit to the Michaelis-Menten equation
directly. The data from the initial velocity studies of the R268M
showed best fit to the equation for a sequential mechanism,
On-line formulae not verified for accuracy
where v is the initial velocity, V
On-line formulae not verified for accuracy
where K
Dissociation constants were determined from modified
Scatchard plots (38) as described previously for the tight binding of
the coenzymes to WT-hALR2(21, 39) . This method relates
the fractional saturation
On-line formulae not verified for accuracy
where E
On-line formulae not verified for accuracy
to determine the
The expression vector was
engineered to contain a pelB leader sequence upstream of the initial
methionine when cloned into the NcoI site, which allows for
the export of target proteins into the periplasmic space. This was done
to ease purification and decrease the amount of recombinant protein
that could form inclusion bodies. The leader sequence is removed in
vivo by a signal peptidase as the protein is
exported(26, 27) . Purified WT and the R268M mutant were
directly sequenced from the N terminus for 10 cycles, and this
confirmed that the signal peptide was completely removed except for an
alanine residue, which preceded the initial methionine of the hALR2
sequence predicted by its cDNA(2, 28) . A comparison of
kinetic constants for the WT-hALR2 with constants previously published
for recombinant hALR2 (11, 24, 25) showed that
the extra alanine residue did not alter the kinetic properties of the
WT-hALR2.
The final enzyme preparations were homogenous as shown by
SDS-polyacrylamide gel electrophoresis (Fig. 1A, lanes 5 and 6). Western blot analysis using purified anti-hALR2
antiserum showed a cross-reacting band in an extract of BL21(DE3)
culture that contained the hALR2/pET20b expression vector and that was
induced with IPTG (Fig. 1B, lane 4). No cross-reactivity
was detected in either the uninduced culture (Fig. 1B, lane
3) or BL21(DE3) cells lacking the expression vector, but induced
by IPTG (Fig. 1B, lane 2). Purified recombinant WT-hALR2
and mutant R268M-hALR2 both cross-reacted with the antiserum.
The introduction of the R268M mutant of hALR2
did not appear to cause any gross structural perturbations. At 22 and
37 °C, both enzymes were stable over 4 h in either the absence or
presence of NADPH/NADH (data not shown). The structural perturbation
introduced in the R268M mutant of hALR2 is essentially only the removal
of the guanidine group of Arg-268, since the
-CH
Product inhibition studies confirmed
that the WT-hALR2 followed an ordered, sequential mechanism.
NADP
Since the mutation had not affected the kinetic mechanism, it was
possible to obtain and compare the kinetic constants k
The kinetic results of WT and R268M-hALR2
showed that there was a 36-fold increase in K
The effect of the R268M mutation on the K
The effect of the mutation on k
The binding
affinity of coenzyme to ALR2 has been shown to be dependent on the
rates of nucleotide binding and release, as well as on the rate of
isomerization of the enzyme-coenzyme complex. The fact that there was a
large increase in the binding of coenzyme (specifically
NADP
The destabilization in
coenzyme binding energy for the removal of Arg-268 for NADH (i.e. in the absence of the 2`-phosphate of NADP(H)) was 0.75 kcal/mol.
As expected, this is much smaller than the 3.2 kcal/mol of binding
energy observed for Arg-268 and NADPH (i.e. when the
2`-phosphate group was present). However, this does indicate that
Arg-268 interacted with other parts of the coenzyme, besides the
2`-phosphate. X-ray crystallography data have demonstrated that Arg-268
forms a hydrogen bond with the oxygen of the adenosine ribose of
NADP(H)(14) , and therefore, the binding energy observed in the
absence of Arg-268 and the 2`-phosphate of the coenzyme quantitatively
describes that interaction.
Grimshaw (5) has demonstrated
that, due to the broad substrate (aldehyde) specificity shown by ALR2,
any transition state stabilization derived from the nonreacting
portions of the substrate must be small. This is probably especially
true for the small aldose sugar DL-glyceraldehyde. The
three-dimensional structure of hALR2 complexed with NADPH describes the
aldehyde binding site as being very hydrophobic, and it is an unlikely
site for the aldose sugar substrate(14) . Therefore, ALR2 must
obtain most of its transition state stabilization energy from its
interaction with coenzyme (5) during the reduction of DL-glyceraldehyde.
The activation energies were calculated
from the rate constants of the binary complexes reacting with DL-glyceraldehyde to give products, i.e. (k
On-line formulae not verified for accuracy
where k
Since the interaction of the 2`-phosphate of NADPH with Arg-268 was
not enhanced during transition state stabilization, other residues that
interact with the 2`-phosphate must be responsible for this
stabilization. One obvious candidate is Lys-262, which is known to form
a salt-link with the negatively charged phosphate of the coenzyme. Two
previous site-directed mutagenesis studies have been done with K262M
mutants (Ref. 24 and also referred to as K263M by Yamaoka et
al.(25) ). When we calculated the extent of destabilization
that removal of Lys-262 has on the transition state, we find that these
studies indicate a destabilization of 1.6 kcal/mol (25) and 1.7
kcal/mol(24) . With the removal of the 2`-phosphate of NADPH
itself causing a total of 2.0 kcal/mol destabilization (I), this, therefore, indicates that, unlike Arg-268, the
interaction of Lys-262 with the 2`-phosphate of NADPH must play a major
role in both coenzyme-enzyme interaction and transition state
stabilization.
In conclusion, we have shown that the 2`-phosphate of
NADP(H) plays a major role in tight coenzyme binding, as well as a
modest role in stabilization of the transition state complexes during
aldehyde reduction by hALR2. Arg-268 contributes significantly to the
binding energy of the coenzyme through its interaction with the
2`-phosphate. Interestingly, a closely related enzyme ALR1 apparently
does not share ALR2's ability to use both NADPH and NADH
coenzymes(50) . ALR1 contains the lysyl and arginyl residues
(Lys-263 and Arg-269(2) ) which form salt linkages with the
2`-phosphate of NADPH that create the strong specificity for NADPH over
NADH, as in ALR2(4) . If ALR1 and ALR2 are similar in their
interactions with the 2`-phosphate of the coenzyme, why, therefore, can
ALR2 use NADH as a coenzyme while ALR1 apparently cannot? It is very
likely that it is due to the same factors that allow NADPH to enjoy a
stronger affinity to ALR2 than to ALR1 (51). That is, interactions of
these two related enzymes differ in other region(s) of the nucleotide
coenzyme such that,in addition to the large effect in coenzyme
specificity caused by the 2`-phosphate, NADH can no longer bind to
ALR1.
of <1µMversus 1.2 mM respectively). Interactions responsible for this
specificity include salt linkages between the highly conserved residues
Lys-262 and Arg-268, and the 2`-phosphate of NADP(H). Previous studies
show that mutation of Lys-262 results in an increase in the K
for both coenzyme and aldehyde
substrate, as well as in the k
of reduction. The
present study shows that mutation of Arg-268 to methionine results in a
36-fold increase in K
and 205-fold
increase in K
for NADPH, but little
change in K
for DL-glyceraldehyde or in the k
of the
reaction. Calculation of free energy changes show that the 2`-phosphate
of NADPH contributes 4.7 kcal/mol of binding energy to its interaction
with WT-hALR2. For the R268M mutant, the interaction of NADPH was
destabilized by 3.2 kcal/mol, indicating that the mutation decreases
the binding energy of NADPH by 65%. The effect of removing Arg-268 in
the absence of the 2`-phosphate of NADPH was virtually identical to the
destabilization of the activation energy in the absence of the
2`-phosphate itself (1.9 versus 2.0 kcal/mol, respectively).
Therefore, while the 2`-phosphate of the coenzyme plays a role in both
coenzyme binding and transition state stabilization during catalysis,
the role of Arg-268 lies strictly in tighter coenzyme binding.
(
)a primarily
NADPH-dependent oxidoreductase, is a member of the aldo-keto reductase
superfamily of enzymes(1, 2, 3) . The enzyme
exhibits a very broad substrate specificity catalyzing the reduction of
a wide variety of aldehydes to their corresponding alcohols (4) and the prevailing view of its physiological role is that of
detoxification of aldehydes(5) . ALR2 is also the first enzyme
of the polyol pathway where it catalyzes the reduction of D-glucose to D-sorbitol(6) . The accumulation
of sorbitol in cells is believed to contribute to the etiology of
diabetic complications (7, 8) and the development of
aldose reductase inhibitors as drugs has been the focus of much
research. Recent advances in our knowledge of the structure, kinetics,
and mechanism of action of ALR2 enhance the prospects for rational
design of aldose reductase inhibitors.
NADP
complex undergoes a slow
rate-limiting conformational change which allows NADP
to dissociate from the enzyme(9, 10) .
Co-crystallization of human ALR2 with NADPH has revealed that the
coenzyme becomes locked into place by movement of a loop (loop 7) that
folds over the coenzyme(14, 15) . This conformational
change contributes to the very tight binding of the coenzyme (9) which, given the high
[NADPH]/[NADP
] physiological ratio (16) ensures that ALR2 is saturated with NADPH at all times.
This allows the enzyme to be set to act rapidly on aldehydes as they
appear(5) . Interaction of coenzyme with ALR2 is critical for
the binding of aldose reductase
inhibitors(17, 18, 19, 20, 21) ,
especially those developed commercially for the treatment of diabetic
complications (e.g. zopalrestat and
alrestatin(17, 21) ).
for both the coenzyme NADPH and the
substrate DL-glyceraldehyde as well as an increase in the k
of the reaction(24, 25) .
Site-directed Mutagenesis
The WT, recombinant
human muscle ALR2 was over expressed in Escherichia coli (BL21(DE3) cells containing pLysS plasmid) using the plasmid,
pET20b (Novagen, Madison, WI) at the NcoI/EcoRI site.
This vector contains a pelB leader sequence upstream of the NcoI cloning site which is designed for export of target
proteins into the periplasmic space. The leader sequence is removed in vivo by signal peptidase as the protein is
exported(26, 27) . Human fetal muscle ALR2 cDNA in
pGEM-4Z subcloned into the EcoRI site was generously supplied
by Chihiro Nishimura(28) . The cDNA was partially digested by NcoI and EcoRI, and fragments separated by agarose
gel electrophoresis. The DNA band corresponding to 1339 base pairs was
cut from the gel and treated with Gel'ase (New England BioLabs)
following the manufacturers protocol. This fragment was then ligated
into pET20b at the NcoI/EcoRI site.
Expression and Enzyme Purification
Ten-liter
bacterial cultures were grown at 37 °C in LB medium with 50
µg/ml ampicillin and 34 µg/ml chloramphenicol to an absorbance
of 0.6 at 660 nm. IPTG was added to a final concentration of 0.4 mM and the cultures were then incubated overnight at 30 °C. The
cells were then harvested by centrifuging at 4420 g for 15 min. The periplasmic fraction was then prepared as
described previously(26, 29, 30) .
Protein Determination
Both purified WT and R268M
apoenzyme concentrations were determined by UV absorption at 280 nm
using = 53,000 M
cm
(32) . Total protein concentration of
crude protein preparations for the purification table ()
were determined by the Bradford assay (33) using bovine serum
albumin as standard.
Purification of Anti-hALR2 Antibody
Antibodies
against hALR2 were prepared by Affinity Biologicals of Hamilton, ON,
using native, muscle hALR2. To remove anti-E. coli IgG, the
antibody preparation was preadsorbed with an extract of BL21 (DE3)
cells containing the pLysS plasmid(34) . The extract was
prepared by growing the culture to saturation, harvesting the cells by
centrifugation, resuspending the cells in buffer containing 10 mM Tris-HCl, 1 mM EDTA, 25% sucrose, pH 7.5. After a second
centrifugation, the cells were then osmotically shocked by suspending
in ice-cold water, and kept on ice for 30 min. An equal volume of serum
(10 ml) containing the antibody preparation was then incubated with
this extract for 1 h at room temperature with constant stirring, and
centrifuged at 12,100 g for 15 min. This preparation
was stored at 4 °C in the presence of 0.02% sodium azide.
Characterization of Protein Samples by Western
Blot
Fractionation of proteins was carried out by
SDS-polyacrylamide denaturing gel electrophoresis (35) by the
conditions suggested previously(36) . Western blots were carried
out as described by Sambrook et al. (37) overnight at a
constant current of 20 mA onto Immobilon Transfer Membrane (Millipore).
The detection system used was goat anti-rabbit IgG alkaline phosphatase
conjugate from Sigma at a dilution of 1/2000.
Steady-state Kinetics and Analyses
The general
procedures have been described(10) . The standard enzyme
activity assay during purification of the WT-hALR2 enzyme was carried
out in 0.1 M sodium phosphate, 0.4 M ammonium sulfate
buffer, pH 7.0, containing 4 mMDL-glyceraldehyde and
100 µM NADPH in a total of 1 ml. The enzyme activity assay
was identical for the R268M mutant, except no ammonium sulfate was
present in the reaction mixture.
is the maximum velocity at saturating substrate concentrations, A and B are the two substrate concentrations, K
and K
are
their corresponding Michaelis constants, and K
is the dissociation constant of substrate A. The K
values in determined from
product inhibition studies were obtained from a fit to Equation 2 for
competitive inhibition,
is the competitive
inhibition constant, I is the inhibitor (NADP
or
NAD
) concentration, and A is the
concentration of the varied substrate (NADPH).
Fluorescence Titration to Determine Coenzyme
Binding
The interaction of NADPH and NADP to
WT-hALR2 was examined by intrinsic enzyme fluorescence under identical
conditions to thoses described previously(10) . The fractional
saturation (
) by coenzyme of the total coenzyme binding sites was
equated to the ratio
F/
F
,
where
F is the amount of fluorescence reduction at a
specified coenzyme concentration and
F
is
the amount of fluorescence reduction at fully saturating coenzyme
concentration.
to the total concentration of ligand, L
, by the equation,
is the total active site
concentration, and K
is the dissociation
constant. A plot of 1/(1-
) versusL
/
yields a theoretically straight line
for a homogenous ligand receptor, with the slope equal to the binding
constant and y axis intercept equal to the concentration of
binding sites. As Stinson and Holbrook noted(38) , it is
imperative that the true
F
be determined.
An error in
F
as little as 5% leads to
nonlinearity (previously attributed to heterogeneity(21) ) for
the plot described by Equation 3. Therefore, the titration curve was
initially fitted to the equation,
F
at infinite
coenzyme concentration, and subsequently used in Equation 3, for the
determination of the true K
.
Purification and Characterization of Wild Type and
Mutant hALR2
summarizes the results of a typical
purification of WT-hALR2 extracted from E. coli BL21(DE3)
cells containing the pLysS plasmid as well as the pET20b/hALR2
expression vector. Routinely, 10 liters of culture gave 2.6 mg of
purified protein for both the WT and R268M mutant. The yield of hALR2
was much lower than had been recorded previously from other expression
systems, including another pET
system(24, 28, 30) . It has been speculated
that, for pET21d, some elements may be present that reduce the vectors
transcription efficiency(40) , and it is possible that these
elements may be present in pET20b as well.
Figure 1:
SDS-polyacrylamide gel electrophoresis
and Western blotting of BL21(DE3) cells and purified wild type and
R268M mutant hALR2. A, Coomassie Brilliant Blue R staining of
a 10% acrylamide gel is shown. Lane 1, molecular weight
standards; lane 2, total cellular protein of BL21(DE3) cells
(not containing pET/hALR2) expression vector) induced by 0.4 mM IPTG overnight; lane 3, total cellular protein of
BL21(DE3) cells, containing the pET/hALR2 expression vector, that were
not induced by IPTG; lane 4, total cellular protein of
BL21(DE3) cells containing the pET/hALR2 expression vector induced by
0.4 mM IPTG overnight; lane 5, purified recombinant
hALR2; lane 6, purified hALR2 with R268M mutation. B,
Western blot analysis of an identical gel to A probed with
purified antibody to human psoas muscle ALR2.
During
purification, endogenously bound nucleotides were removed by the
30-90% ammonium sulfate step and subsequent gel filtration. This
step increased the amount of enzyme that bound to the blue dextran
affinity column, since Cibracon blue interacts at the coenzyme binding
site of proteins(41) . Elution of this column with 1 M
salt resulted in protein free of nucleotides as indicated by
spectrophotometric analysis of eluate which showed an absorbance peak
at 280 nm. This allowed for the spectrophotometric quantization of the
enzyme(32) .
CH
SCH
of methionine should be
fairly isosteric with the
-CH
CH
CH
NH
of
arginine(42) , although there probably would be changes in the
solvation state upon the removal of the positively charged arginine
(43).
Functional Characterization of R268M-hALR2 by
Steady-state Kinetics
Double reciprocal plots of initial
velocity versus NADPH concentration at four fixed levels of DL-glyceraldehyde concentrations show intersecting lines for
R268M (Fig. 2A). This pattern is consistent with a
sequential mechanism for R268M-hALR2. The pattern observed for WT-hALR2
consisted of parallel lines which is usually indicative of a double
displacement mechanism (data not shown). However, it was shown
previously that this pattern results from the tight interaction of
NADPH to the enzyme, since the K was
very low(9, 44) , and the term K
K
in
Equation 1 tended to approach zero, creating a tendency for the lines
to show a parallel pattern(44) . The fact that the mutant enzyme
showed converging lines for the initial velocity experiments indicated
that the K
term was no longer as low as
in the case for the WT-hALR2.
Figure 2:
Steady-state kinetic assays of
R268M-hALR2 mutant. Double reciprocal plots of: A, initial
velocities as a function of NADPH concentration, with each line
representing the DL-glyceraldehyde concentrations of 30
µM (), 70 µM (
), 125 µM (▾), and 350 µM (▾); and B,
product inhibition by NADP
with respect to variable
concentrations of NADPH for the reduction of DL-glyceraldehyde. DL-Glyceraldehyde concentration
was held constant at a saturating concentration of 8 mM. The
NADP
concentrations were 0 µM (
), 50
µM (
), 100 µM (
), 200 µM (▾), and 400 µM (
).
Initial velocity experiments were also
carried out with NADH as the variable coenzyme substrate for both the
WT and R268M-hALR2. Not surprisingly, the patterns observed (converging
lines) were consistent with a sequential mechanism for the reduction of DL-glyceraldehyde.
gave a linear competitive inhibition pattern with
NADPH as the variable substrate, and DL-glyceraldehyde
concentration held at a constant and saturating level. In the case of
R268M, both oxidized coenzymes inhibited competitively against their
reduced counterparts. Therefore, it appears that the overall mechanism
for the recombinant WT-hALR2 was Ordered Bi-Bi and that this was not
altered by substitution of the arginine residue to a methionine.
, K
, k
/K
, and K
for both enzymes. The kinetic constants
for WT and R268M are summarized in , along with the x-fold change in these constants as a result of the R268M
mutation. According to the criteria suggested by Tsai and
Yan(45) , when perturbations in kinetic parameters are
5-fold, the mutated residue is considered to be nonessential or
unimportant to catalysis. When perturbations are
20-fold, the
mutated residue is considered to play a functional role provided the
conformation of the mutant enzyme is not significantly perturbed
relative to WT (in the free form and in complexes with substrates).
Changes between 5- and 20-fold should be interpreted in conjunction
with other evidence.
for NADPH. A more direct indication of the effect of the
mutation can be seen by comparing the binding constants of NADPH with
WT and R268M enzymes, where there is an increase of 205-fold due to the
mutation (). Since Arg-268 has been shown to interact with
the 2`-phosphate of NADPH, NADH was used as an alternate substrate to
see what effect the R268M mutation may have had on the coenzyme in the
absence of the 2`-phosphate group. With NADH as coenzyme, little effect
was seen on the K
(2.8 WT) and K
(3.6 WT). Therefore, Arg-268 interacts
specifically with the 2`-phosphate of NADPH, and has little interaction
with the rest of the molecule. Similar results were obtained with the
oxidized coenzymes, NADP
and NAD
.
of the aldehyde substrate, DL-glyceraldehyde was
very modest (4.8 WT). With hALR2 following an ordered reaction
mechanism where NADPH binds first to the enzyme followed by the
reacting aldehyde, the changes of binding to NADPH will affect the
binding of the aldehyde. Therefore, this change in aldehyde K
is most likely completely due to the
altered binding of NADPH.
was small, only a 1.8-fold increase with NADPH
as coenzyme and DL-glyceraldehyde as substrate, and a 1.4-fold
increase with NADH as coenzyme. The rate-limiting step of the reduction
of aldehydes catalyzed by bovine ALR2 and porcine ALR2 has been shown
to be the rate of isomerization of the E
NADP
complex prior to the release of the
coenzyme(9, 10) . The small increase in the overall rate
is probably due to the looser binding of NADP
to the
enzyme, causing a disruption in the interactions that loop 7 makes with
the coenzyme, which is the basis of the conformational
change(14, 15, 46) . This is assuming that the
rate of isomerization did not increase to the extent that other steps
in the reaction pathway (e.g. rate of hydride transfer) would
become rate-limiting. Previous work with the K262M-hALR2 mutant
indicates that this assumption is realistic(24, 47) .
Primary deuterium isotope effects shows that the rate of
coenzyme-enzyme isomerization is still rate-limiting with a rate of
reaction (k
= 0.93 s
with varying [NADPH] and DL-glyceraldehyde as
substrate) essentially identical to the rate obtained with the R268M
mutant (k
= 1.1 s
).
This indicates that the rate of hydride transfer (and other steps) must
be significantly greater than 0.93 s
. To date, among
the mutants of hALR2 that exhibit enzyme activity, only a mutation with
a residue directly involved in proton transfer (Y48H;(12) ) has
resulted in the rate-limiting step of the reaction not being the rate
of E
NADP
isomerization.
) due to the Arg-268 mutation along with the
observation that the rate of isomerization of the E
NADP
complex was not affected to a
large degree indicates that the mutation had exerted most of its
changes directly on the rate of nucleotide binding and/or release.
Thermodynamic Contribution of Arg-268 and the
2`-Phosphate of Coenzyme
It is possible to gain insight into the
strength and specificity of substrate binding to enzyme by examining
the magnitudes of energy involved in noncovalent
interactions(48) . In this study we were interested in the
electrostatic forces between the negatively charged phosphate group of
the coenzyme NADP(H) and the positively charged group of Arg-268. By
using the alternate coenzyme substrate, NAD(H), and comparing these
results to those obtained with NADPH, we were able to investigate the
effect that removing the 2`-phosphate had on coenzyme binding energy as
well as on transition state stabilization. Likewise, substitution of
Arg-268 to a methionine by site-directed mutagenesis allowed us to
investigate the thermodynamic consequences of removing Arg-268 (Fig. 3).
Figure 3:
Thermodynamic box describing the effects
of removing the 2`-phosphate of NADP(H) and Arg-268 of hALR2,
separately and in combination. 2`Pi-Nucl. is NADP(H); HO-Nucl. is NAD(H); and E is hALR2. The change in
binding energy resulting from the perturbation to enzyme or substrate
structure is given by G
,
G
, or
G
for removal of Arg-268,
the 2`-phosphate, or both, respectively,
G
for removal of the
2`-phosphate in the absence of Arg-268, and
G
for removal of Arg-268 in
the absence of the 2`-phosphate. The series of asterisks represent the salt linkage bond between Arg-268 and the
2`-phosphate of NADP(H).
The thermodynamic consequences of removing the
2`-phosphate as well as Arg-268 of ALR2, and all the interactions with
them, are shown in the free energy profile in Fig. 4. This study
demonstrated that the 2`-phosphate of NADPH contributed 4.7 kcal/mol of
binding energy in its interaction with hALR2 (I). When
Arg-268 was not present, as in the case of the R268M mutant, the effect
of removing the 2`-phosphate of NADPH was to destabilize the coenzyme
binding by 2.3 kcal/mol. As expected, this is considerably smaller than
the 4.7 kcal/mol binding energy observed for the 2`-phosphate when
Arg-268 was present. This indicated that Arg-268 did contribute
significantly to the binding energy of NADPH, although there are other
interactions, such as with Lys-262, Ser-263, and
Thr-265(14, 15) , that provide binding energy in the
absence of Arg-268.
Figure 4:
Free energy profile showing the changes in
the free energy (at 25 °C) for the hALR2NADPH and
hALR2
NADP
ground state intermediates, and the
transition state intermediate for hydride transfer. Also included are
the results of removing the 2`-phosphate of the nucleotide coenzyme
(-2`-P
) or the arginine residue from the enzyme, Arg-268
(-R268). E is hALR2. Changes in ground and transition state
binding energies are based on data from Table
III.
Removal of Arg-268 alone from the enzyme leads
to a destabilization in the binding energy of NADPH by 3.2 kcal/mol.
This value for the salt bridge made by Arg-268 of hALR2 is similar to
the values reported for other enzymes: 3.0 kcal/mol for
NADPH-cytochrome P450 oxidoreductase (42), and 2.9 kcal/mol for an
amino group in chymotrypsin(49) . Arg-268 represents only 1 of
many interactions with the 2`-phosphate, and yet removal of it results
in a loss of over 65% of the total binding energy of the 2`-phosphate
group. This estimate is probably an upper limit since removal of
Arg-268 actually disrupts interactions of other residues with the
2`-phosphate group(42) . But, since the effect was so large, it
illustrated that Arg-268 plays an essential role in binding the
2`-phosphate of NADP(H), and helps explain the strong specificity that
hALR2 has for NADPH over NADH(4) .
/K
)
,
as related by the relationship(48) ,
is the Boltzman constant,
and h is Planck's constant. With the WT-hALR2, the
effect of removing the 2`-phosphate of NADPH was to increase the
activation energy by 2.0 kcal/mol. This indicates that the 2`-phosphate
of NADPH is involved in the stabilization of the transition state
during catalysis. However, the effect of removing both the 2`-phosphate
of NADPH and Arg-268 interaction from the enzyme was virtually
identical to removing the 2`-phosphate itself (1.9 versus 2.0
kcal/mol, respectively). Also, with the removal of Arg-268 alone, and
the 2`-phosphate of NADPH present, the effect on the activation energy
was small, only 0.59 kcal/mol. This is practically identical to the
change in activation energy with NADH as coenzyme when Arg-268 is
removed (0.58 kcal/mol). Therefore, while the 2`-phosphate of the
coenzyme itself plays a significant role in both binding and active
site stabilization during catalysis, the role of Arg-268 appears to be
strictly confined to tighter coenzyme binding.
Table: Purification of WT-hALR2 from 10 liters of
culture
Table: Kinetic and inhibition
constants for nicotinamide nucleotides with WT and R268M-hALR2
Table: Thermodynamic effects of removing Arg-268 of
hALR2
-D-galactopyranoside: WT, wild type.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.