Active Site Tyrosine Research Articles

An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.

The two active-site tyrosine residues of the A protein play non-equivalent roles during initiation of rolling circle replication of bacteriophage P2

The A protein of bacteriophage P2 initiates rolling circle DNA replication by a single-stranded cut at the origin. Two well-conserved tyrosine residues, interspaced by three amino acid residues, are required for the cleavage-joining activity of the protein. The functional relationship between these tyrosine residues was investigated by site-directed mutagenesis. We found that the two tyrosine residues located in the presumed catalytic site of P2 A play non-equivalent functional roles. Tyrosine residue 454 is superior in nicking single-stranded DNA compared to tyrosine residue 450, while both could promote joining at equal efficiency. Specific peptide-oligonucleotide adducts after cleavage reaction and protease digestion could be observed for both tyrosine residues. We propose that tyrosine 454 initiates replication and that tyrosine 450 is able to cleave the DNA only when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication. Also, the involvement of divalent cations in the catalytic activity of P2 A was investigated. While the cleavage reaction was strongly discriminating between different divalent cations, primarily prefering magnesium, the joining reaction showed the same efficiency independently of what divalent cation was provided. This phenomenon could reflect conformational changes of the protein upon binding to DNA. Finally, we found that a large part of the C terminus but not the N terminus is dispensable for initiation of replication both in vivo and in vitro.

Farnesol oxidation in insects: evidence that the biosynthesis of insect juvenile hormone is mediated by a specific alcohol oxidase

The oxidation of farnesol to farnesoic acid is a key step in insect juvenile hormone biosynthesis. We herein present preliminary characterization of the enzyme-catalyzed oxidation of farnesol to farnesal in larval corpora allata homogenates of the tobacco hornworm, Manduca sexta. This conversion, which is highly substrate specific, has a K m apparent of 1 μM and a pH optimum between 6 and 7. Results from chemical modification experiments indicate that the enzyme possesses an active site tyrosine residue. Although farnesol oxidation in adult M. sexta corpora allata homogenates was previously identified as being catalyzed by a dehydrogenase, the corresponding conversion in larvae is not effected by the addition of nicotinamide cofactors. Instead, enzymatic activity is slightly enhanced by the addition of FAD, decreases when incubations are performed anaerobically, and is completely inhibited when either sodium dithionite or glucose oxidase is added. Although the effect of various additives suggests that the oxidation of farnesol to farnesal does not require a metal redox center, 1,10-phenanthroline (but not 4,7-phenanthroline) is a weak irreversible inhibitor of farnesol oxidation (IC 50=11 mM). The addition of exogenous metals (Fe 2+, Cu 2+, Ni 2+, and Co 2+) caused differential effects on farnesol metabolism, with Cu 2+ being highly inhibitory. Taken together, this data suggests that the oxidation of farnesol to farnesal in larval corpora allata is mediated by a specific oxygen-dependent enzyme, perhaps a flavin and/or iron-dependent oxidase.

Synthesis and use of DNA containing a 5'-bridging phosphorothioate as a suicide substrate for type I DNA topoisomerases.

AbstractType I DNA topoisomerases change the linking number of supercoiled DNA by carrying out orcheastrated cleavage and ligation reactions involving phosphodiester bonds. Strand cleavage is not the result of phosphodiester hydrolysis, but the result of transesterification of an active-site tyrosine, creating a 3′-DNA covalent intermediate and a 5′-terminus (5′-OH). Strand ligation occurs during a second transesterification event when the 5′-OH displaces the enzyme (see Fig. 1). A change in DNA linking number results when strand unwinding occurs between these cleavage and ligation reactions. Rationale for suicide substrate design. A phosphodiester linkage at the site of strand cleavage/ligation and an active site tyrosine are diagrammed on the left; the cleaved DNA and the 3′-phosphotyrosyl enzyme covalent complex are diagrammed on the right. In the unmodified phosphodiester, X represents oxygen; for the 5′-bridging phosphorothioate (OPS DNA), X represents sulfur. Chiral phosphorothioate diesters, in which one of the nonbridging oxygens is replaced by sulfur (not diagrammed), have been extensively used as mechanistic probes in enzymology and should not be confused with the achiral 5′-bridging phosphorothioate diesters described here. KeywordsStrand CleavageTrityl GroupSuicide SubstratePhenyl Mercuric AcetateRibose SugarThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid–base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR–Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

A Highly Acidic Tyrosine 9 and a Normally Titrating Tyrosine 212 Contribute to the Catalytic Mechanism of Human Glutathione Transferase A4-4

Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pKa of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pKa 7.1. In both cases the pKa was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pKa values ≥ 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.

Mechanisms of DNA topoisomerase I-induced cell killing in the yeast Saccharomyces cerevisiae.

DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a mechanism of transient DNA strand cleavage characterized by the formation of a phosphotyrosyl bond between the DNA end and active site tyrosine. Camptothecin reversibly stabilizes the covalent enzyme-DNA intermediate by inhibiting DNA religation. During S-phase, collisions with advancing replication forks convert these complexes into potentially lethal lesions. To define the DNA damage induced by alterations in Top1p catalysis and the cellular processes that mediate the repair of such lesions, the yeast Saccharomyces cerevisiae was used. Substitution of conserved residues N-terminal to the active site tyrosine (Tyr-727) produced alterations in the camptothecin sensitivity or catalytic cycle of DNA Top1. For example, substituting Ala for Thr-722 in Top1T722A increased the stability of the covalent enzyme DNA intermediate. As with camptothecin, Top1T722A-induced cytotoxicity was ascribed to a reduction in DNA religation. By contrast, enhanced covalent complex formation by Top1N726H resulted from a relative increase in the rate of DNA cleavage. Conditional yeast mutants were also selected that exhibit temperature-sensitive growth only in the presence of the self-poisoning Top1T722A enzyme. Subsequent analyses of these tah mutants identified 9 genes whose function suppresses the cytotoxic action of camptothecin and Top1T722A. These include genes encoding essential DNA replication proteins (CDC45 and DPB11) and proteins involved in SUMO- or ubiquitination (UBC9 and DOA4).

The Complex of DNA Gyrase and Quinolone Drugs on DNA Forms a Barrier to the T7 DNA Polymerase Replication Complex

Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.

NMR structure of oxidized glutaredoxin 3 from Escherichia coli

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.

Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester

DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA-3'-phosphotyrosyl-enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'-pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein-DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.

Catalytic Mechanism of Galactose Oxidase: A Theoretical Study

Density functional methods, alone and together with molecular mechanics, are used to study the catalytic mechanism of galactose oxidase. This enzyme catalyzes the conversion of primary alcohols to the corresponding aldehydes, coupled with reduction of dioxygen to hydrogen peroxide. It is shown that the proposed mechanism for this enzyme is energetically feasible. In particular the barrier for the postulated rate-limiting hydrogen atom transfer between the substrate and the tyrosyl radical, located at equatorial Tyr272, is very plausible. We propose that the radical site, prior to the initial proton transfer step, is located at the axial tyrosine (Tyr495). The radical is transferred to the equatorial tyrosine (Tyr272) simultaneously with the proton transfer. It is, furthermore, argued that the electron transfer from the ketyl radical intermediate to Cu(II) cannot be very exothermic, because this would render the oxygen reduction steps rate-limiting. Finally, the cysteine cross-link on the active site tyrosine is shown to have very minor effects on the energetics of the reaction.

Expression of Human Topoisomerase I with a Partial Deletion of the Linker Region Yields Monomeric and Dimeric Enzymes That Respond Differently to Camptothecin

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.

DNA Recognition, Strand Selectivity, and Cleavage Mode during Integrase Family Site-specific Recombination

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.

Substitutions of Asn-726 in the Active Site of Yeast DNA Topoisomerase I Define Novel Mechanisms of Stabilizing the Covalent Enzyme-DNA Intermediate

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.

Binding of Alkylurea Inhibitors to Epoxide Hydrolase Implicates Active Site Tyrosines in Substrate Activation

The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.

Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage.

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.

Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity.

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.

Glutathione S-Transferase Catalyzes the Isomerization of (R)-2-Hydroxymenthofuran to Mintlactones

(R)-(+)-Menthofuran is the proximate toxic metabolite of pulegone, the major constituent of the pennyroyal oil, that contributes significantly to the hepatotoxicity resulting from ingestion of this folklore abortifacient pennyroyal oil. Recently, menthofuran was shown to be metabolized by cytochrome P450 to form (R)-2-hydroxymenthofuran. In this paper it is demonstrated that glutathione S-transferase (GST) catalyzes the tautomerization of 2-hydroxymenthofuran to mintlactone and isomintlactone, apparently without the formation of stable glutathione (GSH) conjugates. The reaction strictly required GSH; S-methyl GSH, which binds to the active site and leaves the active site Tyr-9 partly ionized, did not support GST-catalyzed isomerization. It was also determined that the tautomerization reaction requires the active site tyrosine, Tyr-9. The rat GSTA1-1 mutant (Y9F), with the active site tyrosine replaced with phenylalanine, demonstrated no catalytic activity. Rat cytosolic GST A1-1, in the presence of GSH, tautomerized 2-hydroxymenthofuran with apparent KM and Vmax values of 110 μM and 190 nmol/min/nmol GST, respectively. However, the site-directed mutant (F220Y), in which Tyr-9 and GSH in the binary complex [GST · GSH] have lower pKas, exhibited KM and Vmax values of 97 μM and 280 nmol/min/nmol GST, respectively. Similarly, human liver cytosol catalyzed the tautomerization of 2-hydroxymenthofuran in a GST-dependent reaction. The mechanism most consistent with the data is a general-base catalyzed isomerization with GS− serving to deprotonate the substrate to initiate the reaction.

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.