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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.     

Design and synthesis of highly potent and selective (2-arylcarbamoyl-phenoxy)-acetic acid inhibitors of aldose reductase for treatment of chronic diabetic complications

Recent efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective (2-arylcarbamoyl-phenoxy)-acetic acid aldose reductase inhibitors. The compound class features a core template that utilizes an intramolecular hydrogen bond to position the key structural elements of the pharmacophore in a conformation, which promotes a high binding affinity. The lead candidate, example 40, 5-fluoro-2-(4-bromo-2-fluoro-benzylthiocarbamoyl)-phenoxyacetic acid, inhibits aldose reductase with an IC(50) of 30 nM, while being 1100 times less active against aldehyde reductase, a related enzyme involved in the detoxification of reactive aldehydes. In addition, example 40 lowers nerve sorbitol levels with an ED(50) of 31 mg/kg/d po in the 4-day STZ-induced diabetic rat model.     

High-resolution crystal structure of aldose reductase complexed with the novel sulfonyl-pyridazinone inhibitor exhibiting an alternative active site anchoring group

The crystal structure of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase, the first enzyme of the polyol pathway, has been determined to 1.43 angstroms and 0.95 angstroms resolution. The ternary complex of inhibitor, cofactor and enzyme has been obtained by soaking of preformed crystals. Supposedly due to low solubility in the crystallisation buffer, in both structures the inhibitor shows reduced occupancy of 74% and 46% population, respectively. The pyridazinone head group of the inhibitor occupies the catalytic site, whereas the chloro-benzofuran moiety penetrates into the opened specificity pocket. The high-resolution structure provides some evidence that the pyridazinone group binds in a negatively charged deprotonated state, whereas the neighbouring His110 residue most likely adopts a neutral uncharged status. Since the latter structure is populated by the ligand to only 46%, a second conformation of the C-terminal ligand-binding region can be detected. This conformation corresponds to the closed state of the specificity pocket when no or only small ligands are bound to aldose reductase. The two conformational states are in good agreement with frames observed along a molecular dynamics trajectory describing the transition from closed to open situation. Accordingly, both geometries, superimposed in the averaged crystal structure, correspond to snapshots of the ligand-bound and the unbound state. Isothermal titration calorimetry has been applied to determine the binding constants of the investigated pyridazinone in comparison to the hydantoin sorbinil and the carboxylate-type inhibitors IDD 594 and tolrestat. The pyridazinone exhibits a binding affinity similar to those of tolrestat and sorbinil, and shows slightly reduced affinity compared to IDD 594. These studies elucidating the binding mode and providing information about protonation states of protein side-chains involved in binding of this novel class of inhibitors establish the platform for further structure-based drug design.     

Interrupting the hydrogen bond network at the active site of human manganese superoxide dismutase.

Histidine 30 in human manganese superoxide dismutase (MnSOD) is located at a site partially exposed to solvent with its side chain participating in a hydrogen-bonded network that includes the active-site residues Tyr(166) and Tyr(34) and extends to the manganese-bound solvent molecule. We have replaced His(30) with a series of amino acids and Tyr(166) with Phe in human MnSOD. The crystal structure of the mutant of MnSOD containing Asn(30) superimposed closely with the wild type, but the side chain of Asn(30) did not participate in the hydrogen-bonded network in the active site. The catalytic activity of a number of mutants with replacements at position 30 and for the mutant containing Phe(166) showed a 10-40-fold decrease in k(cat). This is the same magnitude of decrease in k(cat) obtained with the replacement of Tyr(34) by Phe, suggesting that interrupting the hydrogen-bonded active-site network at any of the sites of these three participants (His(30), Tyr(34), and Tyr(166)) leads to an equivalent decrease in k(cat) and probably less efficient proton transfer to product peroxide. The specific geometry of His(30) on the hydrogen bond network is essential for stability since the disparate mutations H30S, H30A, and H30Q reduce T(m) by similar amounts (10-16 degrees C) compared with wild type.