Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements

Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new hALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm-1/(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein's electrostatic field. Electrostatic field changes as large as -10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed.     

Stereospecificity of the hydrogen transfer catalyzed by human placental aldose reductase.

Placental aldose reductase (EC 1.1.1.21) was incubated with glucose in the presence of [4A-2H] NADPH prepared in the oxidation of [2-2H] isocitrate by isocitrate dehydrogenase (EC 1.1.1.42) or [4B-2H] NADPH prepared in the oxidation of [1-2H] glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The sorbitol formed from [4A-2H] NADPH contained deuterium and from [4B-2H] NADPH it did not. Therefore, aldose reductase in an A-type enzyme.     

New inhibitors of aldose reductase: anti-oximes of aromatic aldehydes

Aldose reductase is an NADPH-dependent enzyme which catalyzes the reduction of glucose to sorbitol. Specific potent inhibitors of aldose reductase are of potential pharmacological use because elevated levels of sorbitol produced by this enzyme in lens, peripheral nerve, retina, and renal glomeruli may be responsible for the pathogenesis associated with chronic diabetes. These inhibitors could also serve as probes of the mechanism of action of aldose reductase. anti-Oximes of aromatic aldehydes (e.g., benzaldoxime and 4-fluorobenzaldoxime) have proved to be effective inhibitors of aldose reductase rivaling pharmacological agents currently used to inhibit this enzyme in vivo. The kinetic patterns of inhibition in which benzyl alcohol is used as the oxidizable substrate suggest that the inhibition is due to the formation of a stable ternary complex composed of aldose reductase, NADP+, and the anti-oxime. Analogus ternary complexes are formed at the active site of horse liver alcohol dehydrogenase which is also inhibited by anti-oximes of efficient substrates.     

Purified rat lens aldose reductase. Polyol production in vitro and its inhibition by aldose reductase inhibitors.

The production of polyols in vitro by highly purified aldose reductase (EC 1.1.1.21) was monitored by g.l.c. In the presence of NADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.     

Structure of a glutathione conjugate bound to the active site of aldose reductase

Aldose reductase (AR) is a monomeric NADPH-dependent oxidoreductase that catalyzes the reduction of aldehydes, ketones, and aldo-sugars. AR has been linked to the development of hyperglycemic injury and is a clinical target for the treatment of secondary diabetic complications. In addition to reducing glucose, AR is key regulator of cell signaling through it's reduction of aldehydes derived from lipoproteins and membrane phospholipids. AR catalyzes the reduction of glutathione conjugates of unsaturated aldehydes with higher catalytic efficiency than free aldehydes. The X-ray structure of human AR holoenzyme in complex with the glutathione analogue S-(1,2-dicarboxyethyl) glutathione (DCEG) was determined at a resolution of 1.94 A. The distal carboxylate group of DCEG's dicarboxyethyl moiety interacted with the conserved AR anion binding site residues Tyr48, His110, and Trp111. The bound DCEG's glutathione backbone adopted the low-energy Y-shape form. The C-terminal carboxylate of DCEG glutathione's glycine formed hydrogen bonds to Leu301 and Ser302, while the remaining interactions between DCEG and AR were hydrophobic, permitting significant flexibility of the AR and glutathione (GS) analogue interaction. The observed conformation and interactions of DCEG with AR were consistent with our previously published molecular dynamics model of glutathionyl-propanal binding to AR. The current structure identifies major interactions of glutathione conjugates with the AR active-site residues.     

In vitro evaluation of 5-arylidene-2-thioxo-4-thiazolidinones active as aldose reductase inhibitors

2-Thioxo-4-thiazolidinone derivatives were evaluated as aldose reductase inhibitors (ARIs) and most of them exhibited good or excellent in vitro efficacy. Out of the tested compounds, most N-unsubstituted analogues were found to possess inhibitory effects at low micromolar doses and two of them exhibited higher potency than sorbinil, used as a reference drug. The insertion of an acetic chain on N-3 of the thiazolidinone scaffold led to analogues with submicromolar affinity for ALR2 and IC₅₀ values very similar to that of epalrestat, the only ARI currently used in therapy.     

Correlation of binding constants and molecular modelling of inhibitors in the active sites of aldose reductase and aldehyde reductase

Aldose reductase (ALR2) belongs to the aldo–keto reductase (AKR) superfamily of enzymes, is the first enzyme involved in the polyol pathway of glucose metabolism and has been linked to the pathologies associated with diabetes. Molecular modelling studies together with binding constant measurements for the four inhibitors Tolrestat, Minalrestat, quercetin and 3,5-dichlorosalicylic acid (DCL) were used to determine the type of inhibition, and correlate inhibitor potency and binding energies of the complexes with ALR2 and the homologous aldehyde reductase (ALR1), another member of the AKR superfamily. Our results show that the four inhibitors follow either uncompetitive or non-competitive inhibition pattern of substrate reduction for ALR1 and ALR2. Overall, there is correlation between the IC₅₀ (concentration giving 50% inhibition) values of the inhibitors for the two enzymes and the binding energies (ΔH) of the enzyme–inhibitor complexes. Additionally, the results agree with the detailed structural information obtained by X-ray crystallography suggesting that the difference in inhibitor binding for the two enzymes is predominantly mediated by non-conserved residues. In particular, Arg312 in ALR1 (missing in ALR2) contributes favourably to the binding of DCL through an electrostatic interaction with the inhibitor’s electronegative halide atom and undergoes a conformational change upon Tolrestat binding. In ALR2, Thr113 (Tyr116 in ALR1) forms electrostatic interactions with the fluorobenzyl moiety of Minalrestat and the 3- and 4-hydroxy groups on the phenyl ring of quercetin. Our modelling studies suggest that Minalrestat’s binding to ALR1 is accompanied by a conformational change including the side chain of Tyr116 to achieve the selectivity for ALR1 over ALR2.     

Structure-based design of inhibitors of human L-xylulose reductase modelled into the active site of the enzyme

The program GRID was used to design potential inhibitors of human L-xylulose reductase based on a model of the holoenzyme in complex with n-butyric acid. The inclusion of phosphate or carboxylate functional groups in the ligand suggested an increase in the net binding energy of the complex up to 2.8- and 4.0-fold, respectively. This study may be useful in the development of potent and specific inhibitors of the enzyme.     

Conformational change of dihydrofolate reductase near the active site after thiol modification: detected by limited proteolysis

Kinetic studies of chicken liver dihydrofolate reductase (CL-DHFR) and Chinese hamster ovary DHFR (CH-DHFR) activated following p-hydroxymercuribenzoate (p-HMB) modification indicate a conformational change at the active site, suggesting a loosening of the enzyme structure upon SH modification. In the present study, limited proteolysis was applied to detect the subtle conformational changes in SH-modified DHFRs. The digested peptide fragments were separated by Tricine SDS-PAGE and sequenced by Edman auto-degradation. The thiol modifier N-iodoacetyl-N'-(5-sulfo-1-nophthyl) ethylenediamine (IAEANS), which activates these DHFRs only weakly, was used as a control. The results of sequencing showed that compared to native enzyme, there is one additional cleavage site near the active site in p-HMB-modified CL-DHFR, two additional sites in p-HMB-modified CH-DHFR, but no additional site for IAEANS-modified DHFRs. These results indicate that activation of DHFRs following thiol modification is accompanied by a conformational change at or near the active site. This subtle change in the active site conformation results in a pronounced change in enzyme activity. This provides further evidence that flexibility at the active site is essential for full expression of enzyme catalytic activity. Comparing results obtained from previous experiments on guanidine- and urea-activated CL-DHFR, this shows that a conformational change near helix(28-39) is sufficient for full activation of DHFR.     

Structure-activity relationships and molecular modelling of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors

The structure-activity relationships (SARs) of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors (ARIs) were extended by varying the substitution pattern on the 5-arylidene moiety and on N-3. In particular, the introduction of an additional aromatic ring or an H-bond donor group on the 5-benzylidene ring enhanced ALR2 inhibitory potency. Moreover, the presence of a carboxylic anionic chain on N-3 was shown to be an important, although not essential, structural requisite to produce high levels of ALR2 inhibition. The length of this carboxylic chain was critical and acetic acids 4 were the most effective inhibitors among the tested derivatives. Molecular docking simulations into the ALR2 active site accorded with the in vitro inhibition data. They allowed the rationalization of the observed SARs and provided a pharmacophoric model for this class of ARIs.     

Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

Kinetic and structural characterization of the glutathione-binding site of aldose 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, and trans,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.     

Studies of the inhibition of aldose reductase: evidence for multiple site binding

1. Comparison of structure-inhibition relationships and kinetic data between the N-[(4-benzoylamino)phenyl]sulfonyl]amino acids (BAPS-amino acids) and phenylsulfonylamino acids (PS-amino acids) suggests that the additional benzoyl moiety present in the BAPS-amino acids enhances inhibition by direct interaction with aldose reductase (EC 1.1.1.21) without altering the mode of interaction with the enzyme. 2. Also the 2-, 3- and 4-nitro regioisomers of BAPS-glycine (NBAPSG) display parallel structure- inhibition relationships with the 2-, 3- and 4-nitrobenzaldehyde substrates and the 2-, 3- and 4-nitroacetophenone competitive inhibitors. 3. Competition studies and multiple inhibition analyses demonstrate that the 4-nitrobenzoyl group of 4-NBAPSG binds at the substrate site of aldose reductase, while the PS-glycine moiety of 4-NBAPSG binds cooperatively at a distinct site.     

Binding and catalysis by yeast aldose reductase: a substrate-analog approach with new aldose derivatives.

5-Deoxy-D-xylofuranose derivatives and a range of new 5,6-dideoxy analogs of D-glucofuranose bearing azido or fluoro substituents were synthesised and employed as substrates of the NADH-dependent aldehyde reduction catalysed by yeast aldose reductase. In terms of catalytic efficiencies, these products proved to be superior to the parent compounds.     

The use of triazine inhibitors in mapping the active site region of Lactobacillus casei dihydrofolate reductase.

A quantitative structure-activity relationship for the inhibition of Lactobacillus casei dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-X-phenyl)-s-triazines has been derived: for 28 congeners. In this expression C is the molar concentration of triazine causing 50% inhibition, π₃ is the hydrophobic constant for the 3-X-phenyl substituent, MR′ is the molar refractivity of certain substituents, β is an iteratively derived coefficient, and r is the multiple least squares correlation coefficient. This correlation is quite different from those found with the same type of inhibitors acting on bovine and rat liver dihydrofolate reductase. The differences are discussed. The correlation equations for the triazines acting on purified enzymes are compared with equations correlating triazines inhibiting the growth of Staphylococcus aureus and Escherichia coli.     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k(cat)=142 min(-1) and k(cat)/K(m)=1.8x10(7) M(-1) min(-1)). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.