Caccuri, Caccuri - Academia.edu (original) (raw)
Papers by Caccuri, Caccuri
Human Tumor Markers, 1987
Journal of Biological Chemistry, 1991
The reaction of the glutathione transferase from human placenta with a maleimide spin label deriv... more The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.
Giornale italiano di medicina del lavoro ed ergonomia
The interaction among chemicals listed in the Directive CE/39/2000 with skin notation and glutath... more 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 co...
The Italian journal of biochemistry
The binding of the GSH to the GSH transferase pi quenches the protein intrinsic fluorescence more... more The binding of the GSH to the GSH transferase pi quenches the protein intrinsic fluorescence more than the binding of GS-Me. The calculated dissociation constants are 38.6 microM and 90.9 microM for GSH and GS-Me, respectively. From the reported data it is evident that the binding of GSH to GSH transferase pi quenches the intrinsic fluorescence with two different mechanisms. The first one is a conformational change induced by the binding of the GSH and it is present also with the GS-Me binding. A second proposed mechanism is a contact quenching between the sulphydryl GSH group and a tryptophan residue. This suggests that at least one of the tryptophan residues is located near the GSH binding site.
C249 The glutathione S-transferase (GST) family is overexpressed in cancer cells in comparison to... more C249 The glutathione S-transferase (GST) family is overexpressed in cancer cells in comparison to normal tissues and is involved in the mechanism of resistance to several antitumor drugs. To reduce the protective role of GSTs selective inhibitors of the enzyme have been studied. A different class of molecules exploit the GST catalytic power in order to be activated. Among these are: the GSH-peptidomimetic TER 286 (a latent cytotoxin releasing a cyclophosphamide analogue) and brostallicin (a novel drug whose efficacy is enhanced by glutathione (GSH) and/or GST overexpression). Brostallicin is a promising DNA minor groove binding anticancer agent showing preclinical and clinical activity against a variety of human tumors. From a molecular point of view, in vitro experiments strongly support the hypothesis that brostallicin, when activated by GST, reacts with GSH, through a Michael type attack to the double bond, forming a reactive adduct able to bind covalently to the minor groove of DNA. This effect implies that tumor cells expressing relatively high GST/GSH levels are more susceptible to brostallicin antitumor activity. In this work we studied the interaction between brostallicin and two different human GSTs, GSTP1-1 and GSTM2-2. Inhibition experiments indicate that brostallicin binds reversibly to both GSTs and the affinity of brostallicin towards GSTM2-2 is two orders of magnitude higher than that towards GSTP1-1 (Ki values of about 0.3 μM and 40 μM for GSTM2-2 and GSTP1-1, respectively). Bromine appears an essential structural requirement for a tight binding of brostallicin to GSTs. In fact, the debrominated derivative of brostallicin (PNU-230858A) shows very low affinity towards both GSTP1-1 and GSTM2-2. Direct evidence of the involvement of these isoenzymes in the formation of a brostallicin-GSH adduct have been obtained. Reaction products have been separated and characterized by HPLC-mass spectrometry technique. Moreover, the reaction between GSH and brostallicin, has been followed continuously by using a new spectrofluorometric method. Both GSTP1-1 and GSTM2-2 catalyze this reaction however, GSTP1-1 shows a maximum velocity about 4 times higher. The activity of GSTP1-1 reveals a sophisticated allosteric regulation in which transition between two enzyme conformations is triggered by the substrate brostallicin and is modulated by the product of the reaction between GSH and brostallicin. The GSTM2-2 isoenzyme is fully active at brostallicin concentrations comparable to those shown to be effective against different tumor cells. To further corroborate that brostallicin activity is enhanced in cells expressing high GSH/GST levels, studies are ongoing aimed at the evaluation of the activity of brostallicin on GSTπ and GSTμ-transfected versus empty vector-transfected cells. In conclusion, unlike all other cytotoxics and TER 286, brostallicin is the first anticancer agent whose efficacy takes advantage of high intracellular levels of both GSH and GSTs. Considering that the several human tumors display increased levels of GSTP1-1 and/or GSTM2-2 current data support the value of targeted activation of brostallicin in cancer treatment.
European Journal of Biochemistry, 1988
An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named k... more An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.
European Journal of Biochemistry, 1990
To verify a possible involvement of glutathione transferase n in intracellular transport of hemin... more To verify a possible involvement of glutathione transferase n in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques : intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase n was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 pM and the inhibition appeared to be non-competitive with respect to l-chloro-2,4-dinitrobenzene. CD spectra of the bilirubinglutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase n in the hemin transport.
European Journal of Biochemistry, 1993
Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyreny1)maleimide... more Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyreny1)maleimide which modified 1 mol thiol residuelmol protein. The enzyme activity increased about tenfold after the binding. The pyrene-labeled microsomal glutathione S-transferase exibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S-transferase, to larger aggregates. At 25°C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 pM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N-(1-pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.
Neurochemistry International, 1989
A transaminase acting on cystathionine, S-aminoethylcysteine and glutamine has been purified to h... more A transaminase acting on cystathionine, S-aminoethylcysteine and glutamine has been purified to homogeneity from bovine brain by ammonium sulfate precipitation, DE-52 chromatography, octyl-Sepharose chromatography, hydroxylapatite chromatography and gel filtration. The enzyme was purified 4700 times over the bovine brain homogenate and the overall recovery of the enzyme activity was about 18 %. As demonstrated by polyacrylamide gel electrophoresis under native or denaturing conditions, the enzyme has a molecular mass of I00 kDa and is composed of two subunits with approximately identical weight. A single active peak was obtained at pH = 5.24 by chromatofocusing of a homogeneous enzyme preparation. Km values for S-aminoethylcysteine have been calculated using various ct-keto acids as amino acceptor and Km for glutamine has been determined with ct-keto-7-methiolbutyric acid as cosubstrate. The occurrence of the enzyme activity in some bovine brain regions was also studied.
Journal of Molecular Biology, 1998
Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of... more 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 af®nity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by sitedirected mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, puri®ed, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show k cat 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 af®nity towards GSH is accompanied by an induced positive cooperativity as re¯ected by Hill coef®cients 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.
International Journal of Cancer, 2011
Il Farmaco, 2003
The new GSH analogues H-Glo(-Ser-Gly-OH)-OH (5), its O-benzyl derivative 4, and H-Glo(-Asp-Gly-OH... more 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.
Cellular and Molecular Life Sciences, 2001
Diffusion-controlled enzymes are characterized by second-order rate constants in the range 10(8)-... more 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.
Biochemistry, 1997
The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in che... more The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid. † Financial support from the Anti-Cancer Council of Victoria and the National Research Council ACRO is gratefully acknowledged. A.J.O. is a recipient of a National Health & Medical Research Council Postgraduate Research Scholarship, J.R. is a recipient of a Royal Society Fellowship, and M.W.P. is a Wellcome Australian Senior Research Fellow. ‡ The crystallographic coordinates have been deposited in the Brookhaven Protein Data Bank under the filenames 2GSS and 3GSS.
Biochemistry, 2000
We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 thro... more 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-2,4dinitrobenzene. 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. Glutathione transferases (EC 2.5.1.18) (GSTs) 1 are a family of enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of GSH on the electrophilic center of a number of toxic compounds and xenobiotics (1). These cytosolic enzymes have been grouped into at least nine species-independent classes (Alpha, Beta, Delta, Kappa, Mu, Pi, Sigma, Theta and Zeta) on the basis of their amino acid sequence, substrate specificity, and immunological properties (2-8). For a more detailed review on the molecular properties of GSTs, see ref 9. Human placental glutathione transferase (class Pi) (GST P1-1) (10), a homodimeric protein of about 46 kDa, has been extensively studied in different laboratories because of its potential use as a marker during chemical carcinogenesis (11, 12) and its possible role in the mechanism of cellular multidrug resistance against a number of anti-neoplastic agents (13-15). Recent advances suggest a role for GST P1-1 in the regulation of Jun kinase protein (a stress-activated protein which phosphorylates c-Jun) (16) and as a "tissue" transglutaminase-specific substrate in neural cells committed to apoptosis (17). Crystallographic and site-directed mutagenesis studies have indicated that a number of residues are involved in GSH recognition and binding (18-22). The high-resolution structures of human GST P1-1, complexed with GSH analogues or inhibitors (20, 22-26), also help to define the xenobiotic substrate binding site (H-site). The H-site walls are lined by residues Phe 8, Val 35, Ile 104, and Tyr 108, and the base is formed by Val 10 and Gly 205. Although these residues are hydrophobic, the H-site is
Biochemistry, 1998
The conformation of the bound glutathione (GSH) in the active site of the human glutathione trans... more 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
Biochemical and Biophysical Research Communications, 1994
Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1... more Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1), utilizing 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) as co-substrate, show that the kcat value (approximately equal to 1.2 s-1) is pH-independent between pH 4.0 and 7.0 and is scarcely affected by the nature of the leaving group. The pH profile of kcat/KmNBD-Cl suggests a pKa > or = 6.0 for GSH bound to the enzyme. Pre-steady state experiments demonstrate the presence of a burst-phase in which the conjugation product (or the sigma-complex intermediate) accumulates in an amount stoichiometric to the GST active site concentration. These results indicate that the steady state kinetics of GST P 1-1 with NBD-Cl are independent of the deprotonation of the bound GSH between pH 4.0 and 7.0 because the rate-limiting step is the product release. The occurrence of a fast enzymatic conjugation of GSH with a number of poor substrates or even electrophilic inhibitors of GST, mainly performed in a single turnover reaction, may reveal a further detoxicating role of GST.
Biochemical and Biophysical Research Communications, 1998
Substrate selectivity, among glutathione transferase (GST) isoenzymes, appears to be determined b... more 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.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992
A spin-labelled analogue of glutathionc (sl-glutathione) has been used in order to characterize t... more A spin-labelled analogue of glutathionc (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase ~'. The sl-glutathionc shows a competitive inhibition towards glutathione (K i = 14 p.M). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a K d of 3/.tM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.
Human Tumor Markers, 1987
Journal of Biological Chemistry, 1991
The reaction of the glutathione transferase from human placenta with a maleimide spin label deriv... more The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.
Giornale italiano di medicina del lavoro ed ergonomia
The interaction among chemicals listed in the Directive CE/39/2000 with skin notation and glutath... more 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 co...
The Italian journal of biochemistry
The binding of the GSH to the GSH transferase pi quenches the protein intrinsic fluorescence more... more The binding of the GSH to the GSH transferase pi quenches the protein intrinsic fluorescence more than the binding of GS-Me. The calculated dissociation constants are 38.6 microM and 90.9 microM for GSH and GS-Me, respectively. From the reported data it is evident that the binding of GSH to GSH transferase pi quenches the intrinsic fluorescence with two different mechanisms. The first one is a conformational change induced by the binding of the GSH and it is present also with the GS-Me binding. A second proposed mechanism is a contact quenching between the sulphydryl GSH group and a tryptophan residue. This suggests that at least one of the tryptophan residues is located near the GSH binding site.
C249 The glutathione S-transferase (GST) family is overexpressed in cancer cells in comparison to... more C249 The glutathione S-transferase (GST) family is overexpressed in cancer cells in comparison to normal tissues and is involved in the mechanism of resistance to several antitumor drugs. To reduce the protective role of GSTs selective inhibitors of the enzyme have been studied. A different class of molecules exploit the GST catalytic power in order to be activated. Among these are: the GSH-peptidomimetic TER 286 (a latent cytotoxin releasing a cyclophosphamide analogue) and brostallicin (a novel drug whose efficacy is enhanced by glutathione (GSH) and/or GST overexpression). Brostallicin is a promising DNA minor groove binding anticancer agent showing preclinical and clinical activity against a variety of human tumors. From a molecular point of view, in vitro experiments strongly support the hypothesis that brostallicin, when activated by GST, reacts with GSH, through a Michael type attack to the double bond, forming a reactive adduct able to bind covalently to the minor groove of DNA. This effect implies that tumor cells expressing relatively high GST/GSH levels are more susceptible to brostallicin antitumor activity. In this work we studied the interaction between brostallicin and two different human GSTs, GSTP1-1 and GSTM2-2. Inhibition experiments indicate that brostallicin binds reversibly to both GSTs and the affinity of brostallicin towards GSTM2-2 is two orders of magnitude higher than that towards GSTP1-1 (Ki values of about 0.3 μM and 40 μM for GSTM2-2 and GSTP1-1, respectively). Bromine appears an essential structural requirement for a tight binding of brostallicin to GSTs. In fact, the debrominated derivative of brostallicin (PNU-230858A) shows very low affinity towards both GSTP1-1 and GSTM2-2. Direct evidence of the involvement of these isoenzymes in the formation of a brostallicin-GSH adduct have been obtained. Reaction products have been separated and characterized by HPLC-mass spectrometry technique. Moreover, the reaction between GSH and brostallicin, has been followed continuously by using a new spectrofluorometric method. Both GSTP1-1 and GSTM2-2 catalyze this reaction however, GSTP1-1 shows a maximum velocity about 4 times higher. The activity of GSTP1-1 reveals a sophisticated allosteric regulation in which transition between two enzyme conformations is triggered by the substrate brostallicin and is modulated by the product of the reaction between GSH and brostallicin. The GSTM2-2 isoenzyme is fully active at brostallicin concentrations comparable to those shown to be effective against different tumor cells. To further corroborate that brostallicin activity is enhanced in cells expressing high GSH/GST levels, studies are ongoing aimed at the evaluation of the activity of brostallicin on GSTπ and GSTμ-transfected versus empty vector-transfected cells. In conclusion, unlike all other cytotoxics and TER 286, brostallicin is the first anticancer agent whose efficacy takes advantage of high intracellular levels of both GSH and GSTs. Considering that the several human tumors display increased levels of GSTP1-1 and/or GSTM2-2 current data support the value of targeted activation of brostallicin in cancer treatment.
European Journal of Biochemistry, 1988
An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named k... more An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.
European Journal of Biochemistry, 1990
To verify a possible involvement of glutathione transferase n in intracellular transport of hemin... more To verify a possible involvement of glutathione transferase n in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques : intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase n was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 pM and the inhibition appeared to be non-competitive with respect to l-chloro-2,4-dinitrobenzene. CD spectra of the bilirubinglutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase n in the hemin transport.
European Journal of Biochemistry, 1993
Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyreny1)maleimide... more Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyreny1)maleimide which modified 1 mol thiol residuelmol protein. The enzyme activity increased about tenfold after the binding. The pyrene-labeled microsomal glutathione S-transferase exibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S-transferase, to larger aggregates. At 25°C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 pM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N-(1-pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.
Neurochemistry International, 1989
A transaminase acting on cystathionine, S-aminoethylcysteine and glutamine has been purified to h... more A transaminase acting on cystathionine, S-aminoethylcysteine and glutamine has been purified to homogeneity from bovine brain by ammonium sulfate precipitation, DE-52 chromatography, octyl-Sepharose chromatography, hydroxylapatite chromatography and gel filtration. The enzyme was purified 4700 times over the bovine brain homogenate and the overall recovery of the enzyme activity was about 18 %. As demonstrated by polyacrylamide gel electrophoresis under native or denaturing conditions, the enzyme has a molecular mass of I00 kDa and is composed of two subunits with approximately identical weight. A single active peak was obtained at pH = 5.24 by chromatofocusing of a homogeneous enzyme preparation. Km values for S-aminoethylcysteine have been calculated using various ct-keto acids as amino acceptor and Km for glutamine has been determined with ct-keto-7-methiolbutyric acid as cosubstrate. The occurrence of the enzyme activity in some bovine brain regions was also studied.
Journal of Molecular Biology, 1998
Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of... more 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 af®nity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by sitedirected mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, puri®ed, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show k cat 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 af®nity towards GSH is accompanied by an induced positive cooperativity as re¯ected by Hill coef®cients 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.
International Journal of Cancer, 2011
Il Farmaco, 2003
The new GSH analogues H-Glo(-Ser-Gly-OH)-OH (5), its O-benzyl derivative 4, and H-Glo(-Asp-Gly-OH... more 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.
Cellular and Molecular Life Sciences, 2001
Diffusion-controlled enzymes are characterized by second-order rate constants in the range 10(8)-... more 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.
Biochemistry, 1997
The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in che... more The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid. † Financial support from the Anti-Cancer Council of Victoria and the National Research Council ACRO is gratefully acknowledged. A.J.O. is a recipient of a National Health & Medical Research Council Postgraduate Research Scholarship, J.R. is a recipient of a Royal Society Fellowship, and M.W.P. is a Wellcome Australian Senior Research Fellow. ‡ The crystallographic coordinates have been deposited in the Brookhaven Protein Data Bank under the filenames 2GSS and 3GSS.
Biochemistry, 2000
We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 thro... more 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-2,4dinitrobenzene. 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. Glutathione transferases (EC 2.5.1.18) (GSTs) 1 are a family of enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of GSH on the electrophilic center of a number of toxic compounds and xenobiotics (1). These cytosolic enzymes have been grouped into at least nine species-independent classes (Alpha, Beta, Delta, Kappa, Mu, Pi, Sigma, Theta and Zeta) on the basis of their amino acid sequence, substrate specificity, and immunological properties (2-8). For a more detailed review on the molecular properties of GSTs, see ref 9. Human placental glutathione transferase (class Pi) (GST P1-1) (10), a homodimeric protein of about 46 kDa, has been extensively studied in different laboratories because of its potential use as a marker during chemical carcinogenesis (11, 12) and its possible role in the mechanism of cellular multidrug resistance against a number of anti-neoplastic agents (13-15). Recent advances suggest a role for GST P1-1 in the regulation of Jun kinase protein (a stress-activated protein which phosphorylates c-Jun) (16) and as a "tissue" transglutaminase-specific substrate in neural cells committed to apoptosis (17). Crystallographic and site-directed mutagenesis studies have indicated that a number of residues are involved in GSH recognition and binding (18-22). The high-resolution structures of human GST P1-1, complexed with GSH analogues or inhibitors (20, 22-26), also help to define the xenobiotic substrate binding site (H-site). The H-site walls are lined by residues Phe 8, Val 35, Ile 104, and Tyr 108, and the base is formed by Val 10 and Gly 205. Although these residues are hydrophobic, the H-site is
Biochemistry, 1998
The conformation of the bound glutathione (GSH) in the active site of the human glutathione trans... more 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
Biochemical and Biophysical Research Communications, 1994
Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1... more Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1), utilizing 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) as co-substrate, show that the kcat value (approximately equal to 1.2 s-1) is pH-independent between pH 4.0 and 7.0 and is scarcely affected by the nature of the leaving group. The pH profile of kcat/KmNBD-Cl suggests a pKa > or = 6.0 for GSH bound to the enzyme. Pre-steady state experiments demonstrate the presence of a burst-phase in which the conjugation product (or the sigma-complex intermediate) accumulates in an amount stoichiometric to the GST active site concentration. These results indicate that the steady state kinetics of GST P 1-1 with NBD-Cl are independent of the deprotonation of the bound GSH between pH 4.0 and 7.0 because the rate-limiting step is the product release. The occurrence of a fast enzymatic conjugation of GSH with a number of poor substrates or even electrophilic inhibitors of GST, mainly performed in a single turnover reaction, may reveal a further detoxicating role of GST.
Biochemical and Biophysical Research Communications, 1998
Substrate selectivity, among glutathione transferase (GST) isoenzymes, appears to be determined b... more 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.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992
A spin-labelled analogue of glutathionc (sl-glutathione) has been used in order to characterize t... more A spin-labelled analogue of glutathionc (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase ~'. The sl-glutathionc shows a competitive inhibition towards glutathione (K i = 14 p.M). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a K d of 3/.tM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.