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• W2063018977 abstract "Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893–10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, andtrans,trans-2,4-decadienal was 4–1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase. Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893–10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, andtrans,trans-2,4-decadienal was 4–1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase. aldose reductase dl-dithiothreitol N-ethylmaleimide reduced glutathione glutathionyl high pressure liquid chromatography electrospray ionization/mass spectrometry glutathioneS-transferase Aldose reductase (AR)1is an NADPH-dependent aldo-keto reductase that catalyzes the reduction of a wide variety of aldehydes, including glucose (for review, see Ref. 1.Bhatnagar A. Srivastava S.K. Biochem. Med. Metab. Biol. 1992; 48: 91-121Crossref PubMed Scopus (147) Google Scholar). Several lines of evidence indicate that increased flux of hexoses via the AR-catalyzed pathway is one of the underlying causes of tissue injury and dysfunction associated with hyperglycemic states such as diabetes mellitus and galactosemia. In experimental models of diabetes and galactosemia, pharmacological inhibition of AR attenuates, prevents, and/or delays several pleiotropic complications (1.Bhatnagar A. Srivastava S.K. Biochem. Med. Metab. Biol. 1992; 48: 91-121Crossref PubMed Scopus (147) Google Scholar, 2.Yabe-Nishimura C. Pharmacol. Rev. 1998; 31: 21-33Google Scholar). Conversely, in transgenic animals, lens-specific up-regulation of AR accelerates sugar cataract (3.Lee A.Y. Chung S.K. Chung S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2780-2784Crossref PubMed Scopus (287) Google Scholar), providing evidence for a critical role of this enzyme in the genesis of hyperglycemic injury. Nonetheless, clinical trials with AR inhibitors have yielded uncertain results (4.Kador P.F. Robison Jr., W.G. Kinoshita J.H. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 691-714Crossref PubMed Google Scholar, 5.Sarges R. Oates P.J. Prog. Drug Res. 1993; 40: 99-161PubMed Google Scholar), and the long-term efficacy of these drugs in treating diabetic complications remains to be demonstrated. Although the variable results obtained in clinical trials of AR inhibitors may be due, in part, to their poor specificity and an inappropriate dosing schedule, a major impediment to their long-term clinical acceptance relates to a lack of understanding of the normal physiological role of AR. Recent evidence suggests that AR and its isoforms may be involved in several physiological functions such as cell growth and/or differentiation. The expression of AR as well as a similar murine aldo-keto reductase, FR-1 (fibroblast growth factor-regulated protein-1), is enhanced by growth factors such as fibroblast growth factor and epidermal growth factor (6.Jacquin-Becker C. Labourdette G. Glia. 1997; 20: 135-144Crossref PubMed Scopus (27) Google Scholar, 7.Donohue P.J. Alberts G.F. Hampton B.S. Winkles J.A. J. Biol. Chem. 1994; 269: 8604-8609Abstract Full Text PDF PubMed Google Scholar, 8.Hsu D.K. Guo Y. Peifley K.A. Winkles J.A. Biochem. J. 1997; 328: 593-598Crossref PubMed Scopus (26) Google Scholar). Moreover, an AR-related protein is one of the most prominent antigens up-regulated during hepatocarcinogenesis (9.Zeindl-Eberhart E. Jungblut P.R. Otto A. Rabes H.M. J. Biol. Chem. 1994; 269: 14589-14594Abstract Full Text PDF PubMed Google Scholar). In addition, AR may be involved in several cell type-specific functions such as osmoregulation (10.Burg M.B. Am. J. Physiol. 1995; 268: F983-F996Crossref PubMed Google Scholar), fructose (11.Hers H.G. Biochim. Biophys. Acta. 1956; 22: 202-203Crossref PubMed Scopus (132) Google Scholar) and tetrahydrobioprotein (12.Park Y.S. Heizmann C.W. Wermuth B. Levine R.A. Steinerstauch P. Guzman J. Blau N. Biochem. Biophys. Res. Commun. 1991; 175: 738-744Crossref PubMed Scopus (73) Google Scholar) synthesis, and the metabolism of corticosteroids (13.Wermuth B. Monder C. Eur. J. Biochem. 1983; 131: 423-426Crossref PubMed Scopus (73) Google Scholar). However, the most general role of AR may be detoxification of reactive aldehydes. Both AR and FR-1 display high catalytic efficiency with unbranched saturated and unsaturated aldehydes (14.Srivastava S. Chandra A. Bhatnagar A. Srivastava S.K. Ansari N.H. Biochem. Biophys. Res. Commun. 1995; 217: 741-746Crossref PubMed Scopus (168) Google Scholar, 15.Vander Jagt D.L. Kolb N.S. Vander Jagt T.J. Chino J. Martinez F.J. Hunsaker L.A. Royer R.E. Biochim. Biophys. Acta. 1995; 1249: 117-126Crossref PubMed Scopus (187) Google Scholar, 16.Srivastava S. Harter T.M. Chandra A. Bhatnagar A. Srivastava S.K. Petrash J.M. Biochemistry. 1998; 37: 12909-12917Crossref PubMed Scopus (43) Google Scholar, 17.Srivastava S. Watowich S.J. Petrash J.M. Srivastava S.K. Bhatnagar A. Biochemistry. 1999; 38: 42-54Crossref PubMed Scopus (153) Google Scholar). Since such aldehydes are the major bioactive end products of lipid peroxidation (18.Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5903) Google Scholar) and reduction diminishes their bioactivity, these enzymes may be important components of the cellular antioxidant defenses. In vitro studies indicate that the high efficiency of AR in catalyzing the reduction of lipid-derived aldehydes is, in part, due to the high hydrophobicity of the enzyme's active site, which interacts favorably with long alkyl chains (17.Srivastava S. Watowich S.J. Petrash J.M. Srivastava S.K. Bhatnagar A. Biochemistry. 1999; 38: 42-54Crossref PubMed Scopus (153) Google Scholar). Furthermore, because the active site residues of AR do not form extensive hydrogen bonds with the substrates, the enzyme appears to be ill-suited for the reduction of hydrophilic aldehydes such as glucose. These characteristics of the enzyme suggest that a more general role of AR may be reduction of medium- to long-chain aldehydes. However, in most cells, reduction to alcohol is a minor metabolic fate of aldehydes which are readily oxidized to acids. For instance, oxidation to 4-hydroxynonanoic acid accounts for 40–50% of the metabolism of 4-hydroxy-trans-2-nonenal, whereas the alcohol 1,4-dihydroxynonene represents only a minor (<10%) component (18.Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5903) Google Scholar,19.Srivastava S. Chandra A. Wang L. Seifert Jr., W.E. DaGue B.B. Ansari N.H. Srivastava S.K. Bhatnagar A. J. Biol. Chem. 1998; 273: 10893-10900Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Additionally, unsaturated aldehydes, because of their high electrophilicity, readily form covalent adducts with glutathione. As a result, in most glutathione-competent cells, unsaturated aldehydes generated by lipid peroxidation are likely to be readily conjugated with glutathione, and aldehyde oxidoreductases are likely to be presented with an glutathione-aldehyde conjugate, rather than the free aldehyde. Indeed, our studies show that in addition to reducing 4-hydroxy-trans-2-nonenal, AR is also an efficient catalyst for the reduction of the glutathione adduct of 4-hydroxy-trans-2-nonenal (14.Srivastava S. Chandra A. Bhatnagar A. Srivastava S.K. Ansari N.H. Biochem. Biophys. Res. Commun. 1995; 217: 741-746Crossref PubMed Scopus (168) Google Scholar, 17.Srivastava S. Watowich S.J. Petrash J.M. Srivastava S.K. Bhatnagar A. Biochemistry. 1999; 38: 42-54Crossref PubMed Scopus (153) Google Scholar). Nonetheless, the specificity of the enzyme for glutathione-aldehyde conjugates of varying structure has not been examined, and the role of glutathione in facilitating aldehyde binding to the active site of AR remains poorly defined. This study was therefore undertaken to identify the kinetic and structural features of AR that determine its interaction with glutathione conjugates. Alkanals and trans-2- andtrans-4-alkenals were purchased from Aldrich. Glutathione,S-alkyl derivatives of glutathione, γGlu-Cys, Cys-Gly, γGlu-Cys-Glu, NADPH, dl-glyceraldehyde,dl-dithiothreitol (DTT), 5,5′-dithiobis(2-nitrobenzoic acid), menadione, N-ethylmaleimide (NEM), and [3H]glutathione were purchased from Sigma. Sephadex G-25 columns (PD-10) were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). All reagents used were of analytical grade. Gly-Cys-Gly and homoglutathione (γGlu-Cys-βAla) were purchased from Bachem. All other peptides were synthesized commercially. Recombinant human placental AR was prepared and purified as described previously (20.Petrash J.M. Harter T.M. Devine C.S. Olins P.O. Bhatnagar A. Liu S.-Q. Srivastava S.K. J. Biol. Chem. 1992; 267: 24833-24840Abstract Full Text PDF PubMed Google Scholar). GSH was incubated with individual compounds at a 1:1 molar ratio in 0.1 mpotassium phosphate (pH 7.4) at room temperature. The reaction was monitored by following the decrease in the λmax of aldehydes. Free GSH content was monitored by 5,5′-dithiobis(2-nitrobenzoic acid). The conjugates were purified by HPLC using a Rainin reverse-phase ODS C18 column (1 × 30 cm) pre-equilibrated with 0.1% trifluoroacetic acid in water (solvent A) at a flow rate of 1 ml/min. Approximately 1 ml of conjugate (1–10 μmol) was applied to the column, and the eluant was monitored at 220 nm. The conjugates were eluted using a gradient consisting of solvent A and solvent B (acetonitrile) at a flow rate of 1 ml/min. The gradient was established such that solvent B reached 10% in 20 min and 25% in 35 min and held at 25% for 30 min. In an additional 10 min, solvent B reached 60%. In this system, the conjugates eluted with a retention time of 30–45 min. The concentration of the glutathione conjugates was determined by amino acid analysis. After lyophilization, the conjugates were hydrolyzed in a vapor-phase automated hydrolyzer (Perkin-Elmer). The resulting amino acids were derivatized with phenyl isothiocyanate to form phenyl isothiocyanate-derivatives, which were extracted and transferred to an on-line 130/A HPLC apparatus for analysis using an Applied Biosystems 420/H PTC amino acid analyzer/hydrolyzer. In each assay, 100–300 pmol (0.1–0.2%) of the sample were analyzed, which is well within the range of sensitivity of the instrument (25 pmol to 2 nmol). The molecular identity of the GS-aldehyde conjugates was established by electrospray mass spectrometry. The electrospray mass spectrometry experiments were performed on a Micromass ZMD single quadrapole electrospray mass spectrometer. The tuning and calibration solution consisted of polypropylene glycol 2000 in water/methanol (50:50, v/v) containing 0.1% acetic acid. Additional calibration aroundm/z 300 was performed by using GSH, which displayed a well resolved peak at m/z 307. The capillary, cone, and extractor were operated at 3.5, 40, and 3 V, respectively, with 40 p.s.i. N2 at a flow rate of 0.5 liter/min. The source block and desolvation temperatures were set at 80 and 200 °C, respectively. Typically, a 10–20 μmsolution of the conjugates was prepared in acetonitrile/water/acetic acid (50:50:0.1, v/v/v) and injected into the ion source of the spectrometer using a Harvard syringe pump at a flow rate of 5–10 μl/min. The mass spectrometer was set to scan fromm/z 100 to m/z 750 with a step size of 0.5, a dwell time of 2 ms, and a scan speed of 45. When dimers of dehydrated ions were observed, the cone voltage and the source block temperature were varied to optimize formation of the parent molecular ion. Before each experiment, stored recombinant AR was reduced by incubation with 0.1 mDTT at 37 °C for 1 h in 0.1 m potassium phosphate (pH 7.0). Excess DTT was removed by gel filtration using a Sephadex G-25 column (PD-10) pre-equilibrated with N2-saturated 0.1m potassium phosphate (pH 7.0) containing 1 mmEDTA. All operations were carried out at 4 °C to prevent oxidation of the enzyme. The DTT-free, reduced enzyme was stored under N2 and used within 1 h. Enzyme activity was measured at room temperature in a 1-ml reaction system containing 0.1 mpotassium phosphate, 1 mm EDTA (pH 7.0), 10 mmglyceraldehyde, and 0.15 mm NADPH. The reaction was monitored by measuring the disappearance of NADPH at 340 nm using a Gilford Response II spectrophotometer or a Varian Cary 100 Bio spectrophotometer. One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of NADPH/min. The control cuvette (blank) contained all components of the reaction mixture except the enzyme. The substrate concentration was varied over a range extending from 0.2 to 5–7 times the K m. Initial velocity was measured at seven to nine different concentrations of each substrate. Individual saturation curves used to obtainV max and K m were fitted to the general Michaelis-Menten equation using a nonlinear iterative fitting program (21.Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1930) Google Scholar) (Equation 1), v=(Vmax·S)/(Km+S)Equation 1 where v is the enzyme velocity and S is the substrate concentration. Data conforming to linear uncompetitive and noncompetitive or competitive inhibition were fitted to Equations Equation 2, Equation 3, Equation 4, respectively, v=(Vmax·S)/(Km+S(1+I/Kii))Equation 2 v=(Vmax·S)/(Kms(1+I/Kis)+S(1+I/Kii))Equation 3 v=(Vmax·S)/(Kms(1+I/Kis)+S)Equation 4 where I is the inhibitor concentration andK ii and K is are the intercept and slope inhibition constants, respectively. In all cases, the best fit to the data was chosen on the basis of the standard error of the fitted parameter and the lowest values of ς, which is defined as sum of the squares of the residuals divided by the degree of freedom,i.e. n − 1, where n represents the number of velocity measurements. Data are expressed as means ± S.E. The structure of GS-propanal was constructed from the coordinates of glutathione (Protein Data Bank code 1gra) (22.Becker K. Savvides S.N. Keese M. Schirmer R.H. Karplus P.A. Nat. Struct. Biol. 1998; 5: 267-271Crossref PubMed Scopus (148) Google Scholar) and 2-cyclopropylmethylenepropanal (Protein Data Bank code 1hrn) (23.Tong L. Pav S. Lamarre D. Pilote L. LaPlante S. Anderson P.C. Jung G. J. Mol. Biol. 1995; 250: 211-222Crossref PubMed Scopus (29) Google Scholar), with the starting conformation of glutathione the same as that in the crystal structure of glutathione reductase. The chemical structure of GS-propanal is shown in Fig. 1. For AR, the 1.76-Å structure complexed with NADP+ and glucose 6-phosphate was used (Protein Data Bank code 2aq) (24.Harrison D.H. Bohren K.M. Ringe D. Petsko G.A. Gabbay K.H. Biochemistry. 1994; 33: 2011-2020Crossref PubMed Scopus (150) Google Scholar) as the starting model. GS-propanal was positioned in the active site of AR using program O (25.Wilson D.K. Tarle I. Petrash J.M. Quiocho F.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9847-9851Crossref PubMed Scopus (207) Google Scholar). The aldehyde moiety of GS-propanal was positioned such that the carbonyl oxygen of the aldehyde is 2.9 Å from both the NE2 atom of His110 and the hydroxyl group of Tyr48and is in the same position as the carboxylate oxygen of zopolrestat (25.Wilson D.K. Tarle I. Petrash J.M. Quiocho F.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9847-9851Crossref PubMed Scopus (207) Google Scholar). This resulted in the carbonyl moiety being parallel to the nicotinamide ring of NADPH. Two distinct starting orientations of GS-propanal, termed orientations 1 and 2, were examined. The propanal group was oriented similarly in both models, and the positioning of the GS-propanal residues γGlu1 and Gly3 was switched in the two models. The energy of the GS-propanal·AR complex was minimized to reduce both steric strain and close van der Waals contacts. Molecular dynamics calculations were then performed for 0.5 ps at 300 K to enable the bound GS-propanal to sample a large part of the conformational space within the AR active site. A 200-step conjugate gradient energy minimization was performed after the molecular dynamics calculation to generate the final models of the GS-propanal·AR complex. All energy minimization and molecular dynamics calculations were performed with the X-PLOR program (50.Brünger A.T. X-PLOR Manual, Version 3.1. Yale University, New Haven, CT1992Google Scholar). Force-field terms for GS-propanal were constructed from analogous amino acids parameters. To maintain the geometry of the catalytic site, the aldehyde moiety of GS-propanal was constrained with weak distance restraints (nuclear Overhauser effect Restraints) to be within 2.9 Å of the hydroxyl oxygen of Tyr48 and the NE2 atom of His110. In the first series of experiments, the effect of glutathione conjugation on the catalytic efficiency of AR in reducing unsaturated aldehydes was examined. For this, glutathione conjugates of acrolein,trans-2-hexenal, trans-2-nonenal, andtrans,trans-2,4-decadienal were prepared by incubating the alkenals with GSH at room temperature and neutral pH. The conjugates formed were purified by HPLC, and their concentration was determined by amino acid analysis. In some experiments, [3H]GSH was used to conjugate alkenals; and after HPLC purification, the concentration of the conjugates was calculated based upon the radioactivity. Upon ESI+/MS, the conjugates displayed intense molecular ions with m/z values corresponding to a 1:1 adduct between glutathione and the alkenal (Table I). Appropriate dilutions of the conjugates were used for the measurement of AR activity. Since conjugation removes unsaturation at C-3, the kinetic constants obtained with the conjugates were compared with the corresponding aldehyde saturated at this position. Under the experimental conditions used, the aldehydes and their conjugates followed Michaelis-Menten kinetics. To ensure that no side reactions or unexpected interactions contaminated the calculated values of the kinetic parameter, the products generated by AR catalysis were examined. For this, the ESI+/MS spectra of the glutathione conjugates before and after reduction by AR were compared. As shown in Fig. 2 for GS-propanal, incubation with AR led to an increase in them/z value of the conjugate from 364.4 to 366.4, consistent with the reduction of the aldehyde to an alcohol. No significant formation of other ions was detected. Single reduction products were also obtained with other glutathione conjugates (data not shown).Table ISteady-state kinetic parameters for aldose reductase-catalyzed reduction of aldehydes and glutathione-aldehyde conjugatesSubstratem/zK mk catk cat/K mμmmin −1min −1μm −1Propanal5120 ± 32028.1 ± 1.70.005 ± 0.0003GS-propanal364.413.2 ± 187.9 ± 8.76.8 ± 0.7Hexanal8.4 ± 123.4 ± 0.82.9 ± 0.1GS-hexanal406.37.2 ± 174.0 ± 6.610.6 ± 1.0Nonanal25.1 ± 736.9 ± 4.61.5 ± 0.2GS-nonanal448.39.3 ± 766.0 ± 8.97.3 ± 0.9trans-4-Decenal44 ± 134.2 ± 4.50.8 ± 0.1GS-trans-4-decenal460.44.2 ± 155.4 ± 9.913.8 ± 2.3The enzyme activity was determined in 0.1 m potassium phosphate, (pH 7.0) using the indicated aldehyde or GS-aldehyde conjugate in the presence of 0.15 mm NADPH. Before measurement of activity, the recombinant enzyme was reduced by DTT and purified over a PD-10 column. Conjugates were prepared by incubating aldehydes and GSH together in 0.1 m potassium phosphate (pH 7.4) and purified by HPLC as described under “Experimental Procedures.” The concentration of the conjugates was determined by amino acid analysis. The m/z values of the glutathione conjugates were determined by ESI+/MS as described under “Experimental Procedures.” Kinetic constants are expressed as means ± S.E. Open table in a new tab The enzyme activity was determined in 0.1 m potassium phosphate, (pH 7.0) using the indicated aldehyde or GS-aldehyde conjugate in the presence of 0.15 mm NADPH. Before measurement of activity, the recombinant enzyme was reduced by DTT and purified over a PD-10 column. Conjugates were prepared by incubating aldehydes and GSH together in 0.1 m potassium phosphate (pH 7.4) and purified by HPLC as described under “Experimental Procedures.” The concentration of the conjugates was determined by amino acid analysis. The m/z values of the glutathione conjugates were determined by ESI+/MS as described under “Experimental Procedures.” Kinetic constants are expressed as means ± S.E. With each of the aldehydes tested, the catalytic efficiency (k cat/K m) was significantly higher for the conjugate than for the corresponding free aldehyde (Table I). The increases in the catalytic efficiency were, however, variable. A greater enhancement (>1000-fold) of the catalytic efficiency was observed with the small-chain aldehyde acrolein than with intermediate-chain aldehydes such as hexenal and nonenal (3.5–5-fold). The medium-chain unsaturated conjugate GS-trans-4-decenal was reduced with a 16-fold greater efficiency than trans-4-decenal. These results suggest that conjugation with glutathione enhances the catalytic efficiency of AR, particularly for small-chain or unsaturated aldehydes. To examine the specificity of glutathione in facilitating the aldehyde binding/catalysis at the active site, we measured the kinetic parameters of AR with conjugates in which the peptide backbone was systematically varied while retaining the Cys–aldehyde bond. For this analysis, propanal adducts were used because GS-propanal displayed the greatest enhancement of catalytic efficiency over propanal. The conjugates were prepared by incubating the indicated thiol peptides with acrolein. The propanal conjugates formed intense molecular ions upon ESI+/MS, which accounted for >90% of the signal. Some of the conjugates examined, e.g. those with cysteine and Cys-Gly, formed 1:2 adducts (two aldehydes conjugated with the amino acid or the peptide). These adducts were not used for further analysis. The remaining conjugates displayed strict 1:1 stoichiometry. Of these, some, e.g. Gly-Cys-Gly and γGlu-Cys-Glu, displayed additional peaks (data not shown) that were assigned to dehydrated forms of the conjugate. However, since no peaks corresponding to 1:2 adducts were observed, these conjugates were tested as potential AR substrates. The m/z values of the parent peptides and synthesized conjugates are listed in Table II.Table IISteady-state kinetic parameters for aldose reductase-catalyzed reduction of peptide-propanal conjugatesPeptidem/zK mk catk cat/K mPeptideConjugateμmmin −1min −1μm −1γGlu-Cys-Gly308.3364.313.0 ± 1.687.9 ± 8.76.6 ± 0.7αGlu-Cys-Gly308.5364.31.5 ± 0.329.3 ± 2.019.5 ± 1.4γ-Aminobutyric acid-Cys-Gly263.1319.210.1 ± 1.032.7 ± 1.63.2 ± 0.2Gly-Cys-Gly236.2292.233.7 ± 7.179.2 ± 8.42.3 ± 0.3γGlu-Cys-Glu379.1435.39.3 ± 1.143.7 ± 2.64.7 ± 0.3γGlu-Cys-Ala322.2378.324.2 ± 3.028.8 ± 1.41.2 ± 0.06Gly-Cys-Glu308.3364.32.6 ± 0.549.6 ± 1.819.1 ± 0.7γGlu-Cys251.2307.33.2 ± 0.244.5 ± 0.813.9 ± 0.2N-Acetyl-Cys-Glu293.2349.11.6 ± 0.318.0 ± 0.711.2 ± 0.5N-Acetyl-Cys-Gly221.1277.31.6 ± 0.316.9 ± 1.410.6 ± 0.9N-Acetyl-Cys163.8220.263.1 ± 1.7154.2 ± 6.20.86 ± 0.1γGlu-Cys-Ala-Gly379.2435.412.7 ± 3.018.3 ± 1.31.4 ± 0.1αGlu-Cys-Ala-Gly379.2435.372.1 ± 14.235.6 ± 3.40.49 ± 0.05αGlu-Ala-Cys-Gly379.0435.329.6 ± 4.327.6 ± 0.90.93 ± 0.03The m/z values of the peptides were determined by ESI+/MS. The peptides were incubated with acrolein; the resultant peptide-propanal conjugates were purified by HPLC, and theirm/z values were determined before measurement of enzyme activity. The enzyme activity was measured in 0.1 mpotassium phosphate (pH 7.0) using the indicated peptide-propanal conjugate in the presence of 0.15 mm NADPH. The recombinant enzyme was reduced by DTT and purified over a PD-10 column before determination. Values are expressed as means ± S.E. Open table in a new tab The m/z values of the peptides were determined by ESI+/MS. The peptides were incubated with acrolein; the resultant peptide-propanal conjugates were purified by HPLC, and theirm/z values were determined before measurement of enzyme activity. The enzyme activity was measured in 0.1 mpotassium phosphate (pH 7.0) using the indicated peptide-propanal conjugate in the presence of 0.15 mm NADPH. The recombinant enzyme was reduced by DTT and purified over a PD-10 column before determination. Values are expressed as means ± S.E. Modification of the N-terminal glutamate (γGlu1) had profound effects on the catalytic efficiency with which the enzyme catalyzed the reduction of the conjugate. As compared with GS-propanal (in which the amide bond is between the γ-carboxyl of glutamate and the amine of cysteine), the propanal conjugate of αGlu-Cys-Gly (in which cysteine is linked to the α-carboxyl of Glu) was reduced with much higher efficiency by the enzyme, indicating sensitivity of the active site to the geometry of the N-terminal amide linkage. However, removal of the γ-carboxyl of Glu or substitution of Glu with Gly diminished catalytic efficiency by 50–65% (compare γ-aminobutyric acid-Cys-Gly and Gly-Cys-Gly with GSH), suggesting that the presence of Glu at the N terminus enhances catalytic efficiency due to specific interaction between the enzyme and the γ-carboxyl group of Glu. The catalytic efficiency was equally sensitive to alterations in C-terminal glycine. Although γGlu-Cys-Glu was reduced with efficiency slightly lower than GSH, substitution of the C-terminal glycine with alanine led to a marked reduction in activity and poor catalytic activity. However, transposition of glycine to the N terminus led to a peptide whose conjugate was more efficiently reduced than GSH, indicating that an α-amide linkage at the N terminus facilitates catalysis. This is consistent with the higher catalytic efficiency observed with αGlu-Cys-Gly than with GSH. Moreover, removal of the C-terminal glycine, which led to a smaller molecule, enhanced catalysis. The high catalytic efficiency observed with dipeptides was comparable when either glycine or glutamate was linked with cysteine (cf. N-acetyl-Cys-Gly and N-acetyl-Cys-Glu), suggesting that smaller molecules with less structural constraints during binding are more efficiently reduced by the enzyme. A further decrease in the length of the peptide backbone led, however, to a sharp decline in the catalytic efficiency, and the smallest adduct,N-acetylcysteine-propanal, was among the poorest substrates tested, indicating that residues linked to cysteine interact specifically with the active site, providing additional anchoring and stabilization. The catalytic efficiency was also decreased upon increasing the size of the peptide backbone. Thus, γGlu-Cys-Ala-Gly was reduced much less efficiently than GSH, and no improvement was evident when the N-terminal glutamate of the tetrapeptide was linked via the α-carboxyl rather than the γ-carboxyl. In contrast to" @default.
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• W2063018977 date "2000-07-01" @default.
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• W2063018977 title "Kinetic and Structural Characterization of the Glutathione-binding Site of Aldose Reductase" @default.
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• W2063018977 doi "https://doi.org/10.1074/jbc.m909235199" @default.
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