Identification of the Binding Site of Methylglyoxal on Glutathione Peroxidase: Methylglyoxal Inhibits Glutathione Peroxidase Activity via Binding to Glutathione Binding Sites Arg 184 and 185

Methylglyoxal (MG), a physiological &#102 -dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272 , 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.     

Identification of the fatty acid binding site on glutathione S-transferase P.

Glutathione S-transferase P (GST-P) bound a series of endogenous fatty acids (C12-C18). To clarify the function and the binding site of the fatty acids, interaction between fatty acids and GST-P was investigated by using 12-(9-anthroyloxy) stearic acid conjugated with Woodward's reagent K. The fluorescence-conjugated fatty acid noncompetitively inhibited GST activity. After GST-P was covalently labeled with the fatty acid, the enzyme was digested with Lysyl Endopeptidase. From the peptide mapping, a single fluorescence-labeled peptide was obtained. By the sequence analysis, the peptide binding fatty acid was determined as the residues of 141-188 from the amino terminus.     

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.     

Construction of the active site of glutathione peroxidase on polymer-based nanoparticles

A new nanoenzyme model with glutathione peroxidase-like active site was constructed on polystyrene nanoparticle (PN1) via microemulsion polymerization. In this model system, two functional monomers were designed: one is a tellurium-containing compound that was introduced on the surface of the nanoparticle and acts as a catalytic center, and the other one is an arginine-containing compound designed as a binding site for the complexation of the carboxyl group of substrate 3-carboxy-4-nitrobenzenethiol (ArSH, 1). As a new glutathione peroxidase (GPx) mimic, it demonstrated excellent catalytic activity and substrate specificity. In ArSH assay system, it was at least 316,000-fold more efficient than PhSeSePh for the reduction of cumene hydroperoxide (CUOOH) by ArSH. In contrast to model PN2, which lacks of substrate binding site, PN1 exhibits an obvious enhancement in catalytic activity. To further promote the catalytic efficiency, a substrate ArSH surface-imprinted nanoenzyme model (I-PN) was developed. By correctly incorporating and positioning the catalytic center tellurium and functional binding factor guanidinium, a continuative activity enhancement of 596,000-fold for the reduction of CUOOH by catalyst I-PN compared with diphenyl diselenide (PhSeSePh) was observed. The results clearly show that polymeric nanoparticle can be developed as an excellent model for combining most of catalytic factors of enzyme into one scaffold.     

Identification of tyrosine 79 in the tocopherol binding site of glutathione S-transferase pi

Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, k(obs), exhibits a nonlinear dependence on reagent concentration, with K(I) = 19 microM and k(max) = 0.158 min(-)(1). Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on k(obs). These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr(79) is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr(79) to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in K(I) values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.     

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.     

Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.     

Effect of sarcolysine on glutathione peroxidase and glutathione reductase activity in sarcoma C-45

A drop of glutathione peroxidase and glutathione reductase activity was revealed in sarcoma C-45 at the period of its most intensive growth. Repeated sarcolysine injections (1.2 mg/kg, intraperitoneally) caused a sharp fall in the activity of both enzymes with a simultaneous reduction of the ratio of glutathione reductase and glutathione peroxidase activities. The important role of the glutathione enzyme redox system in the realization of antitumour action of the chemotherapeutic drugs is supposed.     

Identification of the binding site for plasma prekallikrein in human high molecular weight kininogen. A region from residues 185 to 224 of the kininogen light chain retains full binding activity

We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.     

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.     

Incorporation of glutathione peroxidase active site into polymer based on imprinting strategy

Glutathione peroxidase (GPx, EC 1.11.1.9) is a key enzyme involved in scavenging of reactive oxygen species in biological system. For developing an efficient GPx-like antioxidant, catalytically necessary amino acid derivatives which located near the GPx active center were prepared as functional monomers. Via predetermined imprinting with substrate glutathione (GSH), a polymer-based GPx mimic with a similar structure of catalytic center of natural GPx was developed, and it demonstrated high-catalytic efficiency and substrate specificity. The imprinting polymer (I-PEM) exhibits GPx-like activity about three times higher than that of 2-SeCD, a cyclodextrin-based GPx mimic. The detailed studies on kinetics revealed that not only the substrate binding but also positional arrangement of reacting groups contribute significantly to the catalytic efficiency of the peroxidase model.     

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Peroxynitrite reductase activity of selenoprotein glutathione peroxidase: a computational study

The peroxynitrite reductase activity of selenoprotein glutathione peroxidase (GPx) has been investigated using density functional theory calculations for peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) substrates through two different "oxidation" and "nitration" pathways. In the oxidation pathway for ONOO-, the oxidation of GPx and the subsequent formation of the selenenic acid (E-Se-OH) occur through a concerted mechanism with an energy barrier of 4.7 (3.7) kcal/mol, which is in good agreement with the computed value of 7.1 kcal/mol for the drug ebselen and the experimentally measured barrier of 8.8 kcal/mol for both ebselen and GPx. For ONOOH, the formation of the E-Se-OH prefers a stepwise mechanism with an overall barrier of 6.9 (11.3) kcal/mol, which is 10.2 (11.2) kcal/mol lower than that for hydrogen peroxide (H2O2), indicating that ONOOH is a more efficient substrate for GPx oxidation. It has been demonstrated that the active site Gln83 residue plays a critical role during the oxidation process, which is consistent with the experimental suggestions. The nitration of GPx by ONOOH produces a nitro (E-Se-NO2) product via either of two different mechanisms, isomerization and direct, having almost the same barrier heights. A comparison between the rate-determining barriers of the oxidation and nitration pathways suggests that the oxidation of GPx by ONOOH is more preferable than its nitration. It was also shown that the rate-determining barriers remain the same, 21.5 (25.5) kcal/mol, in the peroxynitrite reductase and peroxidase activities of GPx.     

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.     

Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity

Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.     

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.     

Two substrate binding sites in ascorbate peroxidase: the role of arginine 172

Site-directed mutagenesis has been used to probe the role of Arg172 in ascorbate utilization by ascorbate peroxidase. Arg172 was changed to lysine, glutamine, and asparagine. Although each of these variants retains the ability to utilize guaiacol as a reductant, they exhibit large decreases in their steady-state rates of ascorbate utilization. Spectroscopic, steady-state, and transient-state experiments indicate that these variant proteins are capable of reacting with hydrogen peroxide to form Compound I, but their ability to oxidize ascorbate to form Compound II, and subsequently the resting state, is severely impeded. Results are presented which highlight the importance of Arg172, and a model is proposed to explain its role in ascorbate utilization.