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PubMed2001

Farnesyl transferase inhibitors: a novel targeted tnerapy for cancer.

Activating oncogenic mutations of the RAS gene are common in cancer, occurring in 30% of solid tumours in adults. Inhibitors of the enzyme farnesyl protein transferase prevent a key step in the post-translational processing of the RAS protein, and were developed initially as a therapeutic strategy to inhibit cell signalling in RAS-transformed cells. As more has been learnt about the biological effects of farnesyl transferase inhibitors on cancer cells, it has become increasingly clear that tumours without oncogenic RAS mutations may also be targets for farnesyl transferase inhibitor therapy. Encouraging results from phase I and II clinical trials have emerged, creating both enthusiasm and new challenges for the optimum clinical development of this important new class of anticancer drug.

Alkyl and Aryl Transferases↗
PubMed2004

Effect of phenobarbital on the induction of glutathione S-transferases in rat testis.

Glutathione S-transferases are a family of multifunctional proteins involved in intracellular transport processes and drug detoxication. In rats, these enzymes are dimeric proteins, and exist as cytosolic and microsomal proteins. The affinity purified rat testicular glutathione S-transferases are comprised of four subunits, Yc of alpha class, Yb and Ybeta of mu class and Ydelta of pi class. On chromatofocusing, they were resolved into six anionic and four cationic isozymes. The cationic isozymes were found to be abundant. All these isozymes on electrophoresis were found to contain heteromers except for two isozymes. The expression of individual subunits and their activity was elevated on treatment with multiple doses of phenobarbital in rat testis. Among all of these, according to the immunological studies, Ydelta, a pi class glutathione S-transferase, was induced predominantly in response to phenobarbital.

Animals↗
PubMed2000

Puromycin-rRNA interaction sites at the peptidyl transferase center.

The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.

Base Sequence↗
PubMed1999

UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA.

The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.

Anti-Bacterial Agents↗
PubMed1999

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.

Adenosine↗
PubMed1995

Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods.

The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

Amino Acid Sequence↗
PubMed1992

Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200 microM, with a kmax of 0.111 min-1 and KI = 185 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.

Affinity Labels↗
PubMed2001

Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis.

Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed.

Acyl Coenzyme A↗
PubMed1985

Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae.

A subunit of choleragen and an erythrocyte ADP-ribosyltransferase catalyze the transfer of ADP-ribose from NAD to proteins and low molecular weight guanidino compounds such as arginine. These enzymes also catalyze the hydrolysis of NAD to nicotinamide and ADP-ribose. The kinetic mechanism for both transferases was investigated in the presence and absence of the product inhibitor nicotinamide by using agmatine as the acceptor molecule. To obtain accurate estimates of kinetic parameters, the transferase and glycohydrolase reactions were monitored simultaneously by using [adenine-2,8-3H]NAD and [carbonyl-14C]NAD as tracer compounds. Under optimal conditions for the transferase assay, NAD hydrolysis occurred at less than 5% of the Vmax for ADP-ribosylation; at subsaturating agmatine concentrations, the ratio of NAD hydrolysis to ADP-ribosylation was significantly higher. Binding of either NAD or agmatine resulted in a greater than 70% decrease in affinity for the second substrate. All data were consistent with a rapid equilibrium random sequential mechanism for both enzymes.

ADP Ribose Transferases↗
PubMed2003

Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase.

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.

Alkyl and Aryl Transferases↗
PubMed2003

Programmed delivery of novel functional groups to the alpha class glutathione transferases.

Here we describe a new route to site- and class-specific protein modification that will allow us to create novel functional proteins with artificial chemical groups. Glutathione transferases from the alpha but not the mu, pi, omega, or theta classes can be rapidly and site-specifically acylated with thioesters of glutathione (GS-thioesters) that are similar to compounds that have been demonstrated to occur in vivo. The human isoforms A1-1, A2-2, A3-3, and A4-4 from the alpha class all react with the reagent at a conserved tyrosine residue (Y9) that is crucial in catalysis of detoxication reactions. The yield of modified protein is virtually quantitative in less than 30 min under optimized conditions. The acylated product is stable for more than 24 h at pH 7 and 25 degrees C. The modification is reversible in the presence of excess glutathione, but the labeled protein can be protected by adding S-methylglutathione. The stability of the ester with respect to added glutathione depends on the acyl moiety. The reaction can also take place in Escherichia coli lysates doped with alpha class glutathione transferases. A control substance that lacks the peptidyl backbone required for binding to the glutathione transferases acylates surface-exposed lysines. There is some acyl group specificity since one out of the three different GS-thioesters that we tried was not able to acylate Y9.

Acylation↗
PubMed2004

Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.

Amino Acid Substitution↗
PubMed1996

Involvement of the carboxyl groups of glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.

Amino Acid Sequence↗
PubMed2000

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes.

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.

Adenine Nucleotides↗
PubMedDate not supplied

Glutathione conjugation of aflatoxin B1 exo- and endo-epoxides by rat and human glutathione S-transferases.

Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMedDate not supplied

Glutathione conjugation of the cytostatic drug ifosfamide and the role of human glutathione S-transferases.

Development of drug resistance against alkylating cytostatic drugs has been associated with higher intracellular concentrations of glutathione (GSH) and increased expression of glutathione S-transferase (GST) enzymes. Therefore, enhanced detoxification by the glutathione/glutathione S-transferase pathway has been proposed as a major factor in the development of drug resistance toward alkylating agents. In this paper we describe 31P NMR and HPLC studies on the spontaneous and glutathione S-transferase catalyzed formation of glutathionyl conjugates of two metabolites of ifosfamide, i.e., 4-hydroxyifosfamide and ifosfamide mustard. At 25 degrees C activated ifosfamide (= 4-hydroxyifosfamide + aldoifosfamide) disappeared faster in the presence of a 10-fold excess of GSH (t1/2 = 107 min) compared to incubations without GSH (t1/2 = 266 min). No evidence for the formation of 4-glutathionyl ifosfamide was found. The ultimate alkylating species of ifosfamide is ifosfamide mustard (IM). In the absence of glutathione, the rate constant for the disappearance of the ifosfamide mustard signal at 25 degrees C (pH 7) was 1.98 x 10(-3) min-1 (t1/2 = 350 min). In the presence of a 10-fold molar excess of glutathione, this rate constant was 1.95 x 10(-3) min-1 (t1/2 = 355 min), indicating that the spontaneous formation of an aziridinium ion is the rate-limiting event in the reaction with glutathione. The aziridinium ion formed from IM can deprotonate upon formation, leading to the formation of a (noncharged) aziridine species. This intermediate (N-(2-chloroethyl)-N'-phosphoric acid diamide) was characterized by 31P, 1H, and 13C NMR spectra. When 2 mM ifosfamide mustard was incubated with 1 mM GSH in the presence of 40 microM GST P1-1, the formation of monoglutathionyl ifosfamide mustard was 2.3-fold increased above the spontaneous level. The other major human isoenzymes tested (A1-1, A2-2, and M1a-1a) did not influence the formation of monoglutathionyl ifosfamide mustard. The results of these studies demonstrate that increased levels of GST P1-1 can contribute to an enhanced detoxification of ifosfamide.

Antineoplastic Agents, Alkylating↗
PubMedDate not supplied

Regioselectivity and quantitative structure-activity relationships for the conjugation of a series of fluoronitrobenzenes by purified glutathione S-transferase enzymes from rat and man.

Quantitative structure-activity relationships (QSAR's) are described for the rate of conjugation of a series of fluoronitrobenzenes with cytosolic as well as with two major alpha and mu class enzymes of rat and human liver, viz., glutathione S-transferases (GST) 1-1, 3-3, A1-1, and M1a-1a. For all purified enzymes studied, the natural logarithm of the rate of conversion of the fluoronitrobenzenes correlates with both the calculated reactivity of the fluoronitrobenzenes for an electrophilic attack (i.e., E(LUMO)) and the calculated relative heat of formation for formation of the respective Meisenheimer complex intermediate (delta delta HF). In addition, the regioselectivity of the reaction was determined and compared. The results obtained strongly support the conclusion that chemical reactivity of the fluoronitrobenzenes is the main factor determining the outcomes of their conversion by all glutathione S-transferase enzymes. The regioselectivities vary only a few percent from one enzyme to another, whereas QSAR lines for all purified enzymes are in the same region and run parallel. This indicates that in the overall reaction the nucleophilic attack of the thiolate anion on the fluoronitrobenzenes, leading to formation of the Meisenheimer complex, is the rate-limiting step in the overall catalysis. The fact that chemical reactivity of the fluoronitrobenzenes is the main factor in setting the outcomes of the overall conversion by the different glutathione S-transferase enzymes implies that extrapolation from rat to results of other species including man, and also from one individual to another, must be feasible. That this is actually the case is clearly demonstrated by the results of the present study.

Aged↗
PubMed2001

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide.

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.

Adenine↗