High glucose induces an early and transient cytoprotective autophagy in retinal Müller cells.

PURPOSE: We investigated the autophagic response of rat Müller rMC-1 cells during a short-term high glucose challenge. METHODS: rMC-1 cells were maintained in 5 mM glucose (LG) or exposed to 25 mM glucose (HG). Western blot analysis was used to evaluate the expression levels of markers of autophagy (LC3-II, p62) and glial activation (AQP4), as well as the activation of TRAF2/JNK, ERK and AKT pathways. Autophagic flux assessment was performed using the autophagy inhibitor chloroquine. ROS levels were measured by flow cytometry using dichlorofluorescein diacetate. ERK involvement in autophagy induction was addressed using the ERK inhibitor FR180204. The effect of autophagy inhibition on cell viability was evaluated by SRB assay. RESULTS: Activation of autophagy was observed in the first 2-6 h of HG exposure. This early autophagic response was transient, not accompanied by an increase in AQP4 or in the phospho-activation of JNK, a key mediator of cellular response to oxidative stress, and required ERK activity. Cells exposed to HG had a lower viability upon autophagy inhibition by chloroquine, as compared to those maintained in LG. CONCLUSION: A short-term HG challenge triggers in rMC-1 cells a process improving the ability to cope with stressful conditions, which involves ERK and an early and transient autophagy activation.

The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K(d)=0.3 µM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K(d)≥2.6 µM) or is inactivated by oxidation (Kd=1.7 µM). This indicates that GSTP1-1's TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex's binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1's association with TRAF2 at the cellular level. The TRAF2-GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex's drastic decline observed in cells progressing from the G0/G1 to the S-G2-M phases. Moreover, GSH's intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2-GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents.

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.

[Study on the interaction between the skin detoxifying enzyme glutathione S-transferase and the substances listed in the CE/39/2000 rules with the "skin" annotation, finalized to the biological monitoring of exposed subjects]. / Studio dell'interazione fra l'enzima detossificante cutaneo glutatione S-trasferasi e le sostanze elencate nella normativa CEE/39/2000 con notazione 'skin' finalizzato al monitoraggio biologico di soggetti esposti.

The interaction among chemicals listed in the Directive CE/39/2000 with skin notation and glutathione S-transferase (GSTP1-1) was studied by following two different experimental approaches. The compounds were incubated with the purified GST isoenzyme GSTP1-1 as well as with the human keratinocytes (PR5) selectively expressing GSTP1-1. Some of the molecules affected the enzymatic activity of both the purified and the intracellular GSTP1-1. In particular, 1,2-dichlorobenzene (DCB), ethylbenzene (ETB), cumene, Sulphotep and 2-eptanone (2-EPT) behaved as inhibitors of the purified GSTP1-1 enzyme, with different inhibition properties according to molecular structure. With the exception of Sulphotep showing a Ki value of 0.2 mM, all compounds reported above were characterized by high Ki values (between 2 and 16 mM) and therefore by low affinity towards GSTP1-1. These results make unlikely the use of a biosensor, based on immobilized GSTP1-1, for the detection of these molecules. On the contrary, Sulphotep can be the object of future investigations. It has to be stressed that the above listed compounds were effective on human keratinocytes, at concentrations two order of magnitude lower than that effective on purified GSTP1-1. In particular, cumene and DCB triggered a clear increase of the intracellular GSTP1-1 activity at concentrations lower than 0.1mM. These interesting results let to hypothesize the use of GSTP1-1 present in the keratinocytes as a marker for biological monitoring of workers exposed to these compounds as well as to evaluate the skin permeability of toxic compounds, not yet identified with a skin notation.

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate.

Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-gamma-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC50=0.11+/-0.01 microM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-gamma-(gamma-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the gamma-glutamyl-cysteine peptide bond, has been developed as gamma-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the gamma-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.

Synthesis and activity of novel glutathione analogues containing an urethane backbone linkage.

The new GSH analogues H-Glo(-Ser-Gly-OH)-OH (5), its O-benzyl derivative 4, and H-Glo(-Asp-Gly-OH)-OH (9), characterized by the replacement of central cysteine with either serine or aspartic acid, and containing an urethanic fragment as isosteric substitution of the scissile gamma-glutamylic junction, have been synthesized and characterized. Their ability to inhibit human GST P1-1 (hGST P1-1) in comparison with H-Glu(-Ser-Gly-OH)-OH and H-Glu(-Asp-Gly-OH)-OH, which are potent competitive inhibitors of rat GST 3-3 and 4-4, has been evaluated. In order to further investigate the effect of the isosteric substitution on the binding abilities of the new GSH analogues 4, 5 and 9, the previously reported cysteinyl-containing analogue H-Glo(-Cys-Gly-OH)-OH has been also evaluated as a co-substrate for hGSTP1-1.

Superefficient enzymes.

Diffusion-controlled enzymes are characterized by second-order rate constants in the range 10(8)-10(10) M(-1)s(-1). These values are at the upper end of the observed rates of many enzyme-substrate reactions and have been predicted by theoretical studies on bimolecular reaction in solution. Such enzymes are considered to be perfect, since their rate-limiting step is not due to any chemical event but to the diffusional association rate between the enzyme and the substrate. Often the enzyme-substrate encounter is facilitated either through the presence of a strong attractive electric field, produced by charges on the enzyme surface, or through the reduction in the dimension of the search process. Here we provide a brief review of some of the enzymes characterized by a very fast second-order constant, focusing attention on triose phosphate isomerase and Cu,Zn superoxide dismutase taken as typical examples of such highly tuned enzymes.

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases.

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases.

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.

Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1.

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.

Proton release on binding of glutathione to alpha, Mu and Delta class glutathione transferases.

Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k(1)>/=(2-5)x10(6) M(-1).s(-1)], which is slowly converted into the final Michaelis complex (k(2)=1100-1200 s(-1)). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1-1 (GST P1-1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35 degrees C, glutathione (GSH) binding to GST P1-1 displays positive cooperativity, whereas negative cooperativity occurs below 25 degrees C. This binding mechanism minimizes changes of GSH affinity for GST P1-1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1-1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.

Shifting substrate specificity of human glutathione transferase (from class Pi to class alpha) by a single point mutation.

Substrate selectivity, among glutathione transferase (GST) isoenzymes, appears to be determined by a few residues. As part of study to determine which residues are class-specific determinants, Tyr 108 (an important residue of the class Pi) has been changed to a valine, the structural equivalent of a class Alpha enzyme. Using a panel of selected substrates, "diagnostic" for either class Pi or Alpha, it is shown here that this single mutation significantly alters the catalytic properties of the class Pi enzyme and shifts the substrate specificity of the enzyme toward that of the class Alpha enzyme.

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure.

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.

Proton release upon glutathione binding to glutathione transferase P1-1: kinetic analysis of a multistep glutathione binding process.

The fate of the thiol proton coming from the ionization of the sulfhydryl group of GSH in the active site of glutathione transferase P1-1 has been studied. pH changes caused by the binding of GSH to the enzyme in the absence of any inorganic buffer indicate that the thiol proton leaves the active site when the binary complex is formed. The amount of protons released is stoichiometric to the amount of GSH thiolate formed in the G-site. The apparent pKa value for the bound GSH, calculated with this potentiometric approach, is 6.18 +/- 0.09; very similar values are found by spectrophotometric (6.20 +/- 0.12) and by kinetic (6.00 +/- 0.08) experiments. Binding of S-hexylglutathione does not cause any proton release. Stopped-flow data obtained by means of an acid-base indicator show that the proton extrusion process (apparent t1/2 = 1.1 +/- 0.1 ms at 15 degrees C) is not rate limiting in turnover (apparent t1/2 = 34 +/- 4 ms at 15 degrees C). By comparing the kinetic behavior of three distinct events occurring during the binding of GSH to the enzyme, i. e., proton release, ionization of bound GSH and quenching of intrinsic fluorescence, it appears that the binding process follows a multistep mechanism possibly involving the conformational transition of a weak precomplex into the final Michaelis complex. This step is modulated by helix 2 motions and may be rate limiting at physiological GSH concentrations. These findings, coming from kinetic studies, are consistent with NMR data [Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027] and time-resolved fluorescence experiments [Stella, L., Caccuri, A. M., Rosato, N., Nicotra, M., Lo Bello, M., De Matteis, F., Mazzetti, A. P., Federici, G., and Ricci, G., manuscript in preparation].

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione.

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.

Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on alpha-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.

Inhibition of human placenta glutathione transferase P1-1 by the antibiotic calvatic acid and its diazocyanide analogue--evidence for multiple catalytic intermediates.

The inhibition mechanism of the dimeric human placenta glutathione transferase (GST) P1-1 by calvatic acid and the reaction intermediates, i.e. the diazocyanide analogue of calvatic acid, has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different molar ratios of inhibitor/GST P1-1 indicate that 1 mol calvatic acid inactivates 1 mol GST P1-1, containing two catalytically equivalent active sites. However, 2 mol of the diazocyanide analogue of calvatic acid inactivate 1 mol GST P1-1. Two disulfide bridges/dimer, probably between Cys47 and Cys101, have been formed during the reaction of GST P1-1 with calvatic acid and its diazocyanide analogue. The apparent second-order rate constants for GST P1-1 inactivation by calvatic acid and its diazocyanide analogue are 2.4+/-0.3 M(-1) s(-1) and (8.5+/-0.7) x 10(3) M(-1) s(-1), respectively. The reaction of calvatic acid with free L-cysteine can be described by a simple process with an apparent second-order rate constant of (5.0+/-0.4) x 10(1) M(-1) s(-1). In contrast, a transient species occurs during the reaction of the diazocyanide analogue of calvatic acid with free L-cysteine. Kinetics may be described by a second-order process [the rate constant being (8.0+/-0.5) x 10(3) M(-1) s(-1)] followed by a first-order decay [the rate constant corresponding to (1.2+/-0.1) x 10(1) s(-1)]. Calvatic acid represents an enzyme inhibitor acting much slower than its reaction intermediates (i.e. its diazocyanide analogue).