Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.     

Stereoselectivity of rat liver glutathione transferase isoenzymes for alpha-bromoisovaleric acid and alpha-bromoisovalerylurea enantiomers.

The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.     

Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradation by gamma-glutamyl transpeptidase.

Inhibitors for glutathione S-transferase (GST) iso-enzymes from rat liver with high affinity for the glutathione-binding site (G-site) have been developed. In previous studies, a model was described for the G-site of GST (Adang, A. E. P., Brussee, J., van der Gen, A., and Mulder, G. J. (1990) Biochem. J. 269, 47-54) in terms of essential and nonessential interactions between groups in glutathione (GSH) and the G-site. Based on this model, compounds were designed that have high affinity for the G-site but cannot be conjugated. In the dipeptide gamma-L-glutamyl-D-aminoadipic acid (gamma-L-Glu-D-Aad), the L-cysteinylglycine moiety is replaced by D-aminoadipic acid. This dipeptide is an efficient competitive inhibitor (toward GSH) of mu class GST isoenzymes with Ki values of 34 microM for GST isoenzyme 3-3 and 8 microM for GST isoenzyme 4-4. Other GSH-dependent enzymes, such as gamma-glutamyl transpeptidase (gamma-GT), glutathione reductase, and glutathione peroxidase, were not inhibited by 1 mM of gamma-L-Glu-D-Aad. Inhibition is also highly stereospecific since gamma-L-Glu-L-Aad is only a poor inhibitor (Ki = 430 microM for GST 3-3). Gamma-L-Glutamyl-D-norleucine also had a much higher Ki value for GST 3-3. Thus, the presence of a delta-carboxylate group in D-Aad appears to be essential for a high affinity inhibitor. An additional hydrophobic group did not result in increased inhibitory potency. In a different approach, the gamma-L-glutamyl moiety in GSH was replaced by delta-L-aminoadipic acid; delta-L-Aad-L-Cys-Gly is an efficient cosubstrate analogue for GSTs with Km values comparable to GSH and Vmax values ranging from 0.24 to 57 mumol/min/mg for the different GSTs. The structures of the efficient inhibitor and the cosubstrate analogue were combined in delta-L-Aad-D-Aad, which had a Ki value of 68 microM with GST 3-3. In order to investigate their possible use in vivo studies, the degradation of gamma-L-Glu-D-Aad and delta-L-Aad-L-Cys-Gly by gamma-GT was investigated. The peptides showed no measurable hydrolysis rates under conditions where GSH was rapidly hydrolyzed. Thus, an efficient, mu class-specific GST inhibitor and a gamma-glutamyl-modified cosubstrate analogue of GSH were developed. Their gamma-GT stability offers the possibility to use these peptides in in vivo experiments.     

Ligand-based protein alignment and isozyme specificity of glutathione S-transferase inhibitors

Glutathione S-transferases (GST, E.C.2.5.1.18) comprise a family of detoxification enzymes. Elevated levels of specific GST isozymes in tumor cells are thought responsible for resistance to chemotherapeutics, which renders selective GST inhibitors potentially useful pharmaceutical agents. We discuss the development of a structure activity model that rationalizes the isozyme selectivity observed in a series of 12 glutathione (GSH) analogues. Enzymatic activity data was determined for human P1-1, A1-1, and M2-2 isozymes, and these data were then considered in light of structural features of these three GST proteins. A survey of all GST structures in the PDB revealed that GSH binds to these proteins in a single “bioactive” conformation. To focus on differences between binding sites, we exploited our finding of a common GSH conformation and aligned the GST x-ray structures using bound ligands rather than the backbones of the different proteins. Once aligned, binding site lipophilicity and electrostatic potentials were computed, visualized, and compared. Docking and energy minimization exercises provided additional refinements to a model of selectivity developed initially by visual analysis. Our results suggest that binding site shape and lipophilic character are key determinants of GST isozyme selectivity for close GSH analogues. Proteins 28:202–216, 1997. © 1997 Wiley-Liss Inc.     

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.     

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

Differential scanning fluorometry signatures as indicators of enzyme inhibitor mode of action: case study of glutathione S-transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization.     

Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs

Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.     

Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone

High multiplicity of GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S-transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions.     

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.     

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (pI) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with pI values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with pI values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.     

Purification and characterization of a labile rat glutathione transferase of the Mu class.

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

Thymine hydroperoxide, a substrate for rat Se-dependent glutathione peroxidase and glutathione transferase isoenzymes

The thymine hydroperoxide, 5-hydroperoxymethyluracil, is a substrate for Se-dependent glutathione (GSH) peroxidase and the Se-independent GSH peroxidase activity associated with the GSH transferase fraction. These enzymes may contribute to repair mechanisms for damage caused by oxygen radicals. GSH transferases 1-1, 2-2, 3-3, 4-4, 6-6, and 7-7 [(1984) Biochem. Pharmacol. 33, 2539-2540] are shown to differ considerably in their ability to utilize this substrate. For example, high activity is found in GSH transferase 6-6 which is the major isoenzyme in spermatogenic tubules where DNA synthesis is so active and faithful DNA replication so important. The activity of the purified GSH transferase isoenzymes towards 5-hydroperoxymethyluracil is comparable with their activity towards other endogenous substrates related to cellular peroxidation such as linoleate hydroperoxide and 4-hydroxynon-2-enal or biologically important xenobiotic metabolites such as benzo(a)pyrene-7,8-diol-9,10-oxide.     

5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.     

Synthesis and nucleophilic reactivity of a series of glutathione analogues, modified at the gamma-glutamyl moiety.

A series of GSH analogues with modifications at the gamma-glutamyl moiety was synthesized and purified by following peptide chemistry methodology. Benzyl, benzyloxycarbonyl and t-butyloxycarbonyl protective groups were used to protect individual amino acid functional groups. The formation of peptide bonds was accomplished through coupling of free amino groups with active esters, generated by reaction of the carboxylate functions with dicyclohexylcarbodi-imide and 1-hydroxybenzotriazole. The protecting groups in the tripeptides were removed in a single step by using Na in liquid NH3. Precautions were taken in order to prevent oxidation of the thiol function in the cysteine residue. Thus GSH analogues containing both L- and D-glutamic acid and L- and D-aspartic acid, coupled to cysteinylglycine through both the alpha- and the omega-carboxylate group, were synthesized. Also, decarboxy-GSH and deamino-GSH, lacking one functional group in the glutamate moiety, were prepared. The spontaneous non-enzyme-catalysed nucleophilic reaction of these GSH analogues with the electrophilic model substrate 1-chloro-2,4-dinitrobenzene showed appreciable rate differences, indicating the importance of intramolecular interactions in determining the nucleophilic reactivity of the thiol function in the cysteine residue. In particular, the free amino group in the gamma-L-glutamic acid residue appears to play a crucial role in activating the thiol group in GSH. In an adjacent paper [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] these results are compared with those obtained in a study on the ability of these GSH analogues to act as a co-substrate in the glutathione S-transferase-catalysed conjugation reaction with 1-chloro-2,4-dinitrobenzene.     

Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase.

The role of the hydroxyl group of tyrosine 6 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione S-transferase has been examined by x-ray crystallography and site-specific replacement of the residue with phenylalanine and evaluation of the catalytic properties of the mutant enzyme. This particuar tyrosine residue is conserved in the sequences of all of the cytosolic enzymes and is found, in crystal structures of both isoenzyme 3-3 from the mu-gene class and an isoenzyme from the pi-gene class, to be proximal to the sulfur of glutathione (GSH) or glutathione sulfonate bound at the active site. The 2.2-A structure of the binary complex of isoenzyme 3-3 and GSH indicates that the hydroxyl group of Tyr6 is located 3.2-3.5 A from the sulfur of GSH, well within hydrogen bonding distance. Removal of the hydroxyl group of Tyr6 has essentially no effect on the dissociation constant (22 +/- 3 microM) for GSH. Nevertheless the Y6F mutant exhibits a turnover number which is only about 1% that of the native enzyme when assayed at pH 6.5 with either 1-chloro-2,4-dinitrobenzene (CDNB) or 4-phenyl-3-buten-2-one. UV difference spectra of the binary enzyme-GSH complexes suggest that the predominant ionization state of GSH in the active site of the Y6F mutant is the neutral thiol (e.g. EY6F.GSH) which is in contrast to the native enzyme in which the thiol is substantially deprotonated (e.g. E.GS-). Spectrophotometric titration suggests that the pKa of the thiol is 6.9 +/- 0.3 in the E.GSH complex and greater than or equal to 8 in the EY6F.GSH binary complex. In addition, the pH dependence of kcat/KmCDNB reveals that the reactions catalyzed by the native enzyme and the Y6F mutant are dependent on a single ionization in the E.GSH and EY6F.GSH complexes with pKa = 6.2 +/- 0.1 and 7.8 +/- 0.3, respectively. The results suggest that the hydrogen bond between Tyr6 and the enzyme-bound nucleophile helps to lower the pKa of GSH in the binary enzyme-substrate complex.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.