©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on Human Aldose Reductase
PROBING THE ROLE OF ARGININE 268 BY SITE-DIRECTED MUTAGENESIS (*)

Terrance J. Kubiseski , T. Geoffrey Flynn (§)

From the (1)Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Aldose reductase (ALR2) shows a strong specificity for its nucleotide coenzyme, binding NADPH much more tightly than NADH (K 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.


INTRODUCTION

Aldose reductase (ALR2),()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.

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 enzymeNADP 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) ).

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 for both the coenzyme NADPH and the substrate DL-glyceraldehyde as well as an increase in the k of the reaction(24, 25) .

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.


EXPERIMENTAL PROCEDURES

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.

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.

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) .

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.

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.

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 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,

On-line formulae not verified for accuracy

where K 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.

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 to the total concentration of ligand, L, by the equation,

On-line formulae not verified for accuracy

where E 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,

On-line formulae not verified for accuracy

to determine the F at infinite coenzyme concentration, and subsequently used in Equation 3, for the determination of the true K.


RESULTS AND DISCUSSION

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.

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.


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) .

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 -CHCHSCH of methionine should be fairly isosteric with the -CHCHCHNH 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 KK 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.

Product inhibition studies confirmed that the WT-hALR2 followed an ordered, sequential mechanism. NADP 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.

Since the mutation had not affected the kinetic mechanism, it was possible to obtain and compare the kinetic constants k, 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.

The kinetic results of WT and R268M-hALR2 showed that there was a 36-fold increase in K 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.

The effect of the R268M mutation on the K 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 Kis most likely completely due to the altered binding of NADPH.

The effect of the mutation on k 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 ENADP 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 ENADP isomerization.

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) due to the Arg-268 mutation along with the observation that the rate of isomerization of the ENADP 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 hALR2NADP 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) .

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/K), as related by the relationship(48) ,

On-line formulae not verified for accuracy

where k 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.

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.

  
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



FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada (to T. G. F.), a Medical Research Council Studentship (to T. J. K.), and a Queen's University Faculty of Graduate Studies Bursary (to T. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 613-545-2494; Fax: 613-545-2497; E-mail: flynntg@qucdn.queens.ca.

The abbreviations used are: ALR2, aldose reductase; ALR1, aldehyde reductase; R268M, substitution of arginine 268 with methionine; C298S, substitution of cysteine 298 with serine; K268M, substitution of lysine 262 with methionine; IPTG, isopropyl-1-thio--D-galactopyranoside: WT, wild type.


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