Conformational diversity and enantioconvergence in potato epoxide hydrolase 1.

Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio- and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio- and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

Glutathione transferases with novel active sites isolated by phage display from a library of random mutants.

Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection among variant mutant forms based on specific adsorption to immobilized active-site ligands. A library of mutant enzymes differing in the active-site region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.

Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105.

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library.

A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.

Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene.

Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164(-Gln167) was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.

Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a-1a.

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a-1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme were replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys-deficient mutant forms of glutathione transferase M1a-1a were catalytically active with the tested substrates and their binding of inhibitors, measured by I50, were not significantly different from the values previously obtained for the wild-type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro.

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase. We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by coexpression of molecular chaperonins GroEL/ES.

A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase Dh1A from Xanthobacter autotrophicus strain GJ10 is presented. The strategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble Dh1A. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type Dh1A as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)5-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant Dh1A. The purified His-tagged enzyme, Dh1A-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.

A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes.

His15 is a conserved amino acid residue in all known class Mu glutathione transferases. This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity. The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli. Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized. The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC. For all the mutant forms except H15N, the sp. act. with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity. None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme. The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base. The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation. A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.

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.

Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA.

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.

Contribution of amino acid residue 208 in the hydrophobic binding site to the catalytic mechanism of human glutathione transferase A1-1.

Glutathione transferases (GSTs) catalyze the nucleophilic attack of the thiolate of glutathione on a variety of noxious, often hydrophobic, electrophiles. The interactions responsible for the binding of glutathione have been deduced in great detail from the 3-dimensional structures that have been solved for three different GSTs, each a member of a distinct structural class. However, the interactions of the electrophilic substrates with these enzymes are still largely unexplored. The contribution of the active-site Met208 to aromatic and benzylic chloride substitution reactions catalyzed by human class Alpha GST A1-1 has been evaluated by comparison of wild-type enzyme with variants mutated in position 208. The results show that the amino acid residue at position 208 primarily affects the aromatic substitution reaction, tested with 1-chloro-2,4-dinitrobenzene as substrate, possibly by interacting with the delocalized negative charge of the substituted ring structure in the transition state.

Hydrophobic sequences can substitute for the wild-type N-terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases selection of high-affinity N-terminal region variants by phage display.

A phage-display library of the cysteine-proteinase inhibitor, cystatin A, was constructed in which variants with the four N-terminal amino acids randomly mutated were expressed on the surface of filamenteous phage. Screening of this library for binding to papain gave predominantly variants with a glycine residue in position 4. This finding is in agreement with previous conclusions that glycine in this position is essential for tight binding of cystatin A to cysteine proteinases by allowing optimal interaction of the N-terminal region of the inhibitor with the enzyme. In contrast, the first three residues of the variants obtained by the screening were more variable. Two variants were identified with similar affinities for papain as the wild-type inhibitor, but with these residues, Val-Phe-Thr- or Ile-Leu-Leu, differing appreciably from those of the wild-type, Met-Ile-Pro. Other sequences of the N-terminal region, presumably mainly hydrophobic, can thus substitute for the wild-type sequence and contribute similar energy to the inhibitor-proteinase interaction. The two variants binding tightly to papain differed in their affinity for cathepsin B, demonstrating that cystatin variants with increased selectivity for a particular target cysteine proteinase can be obtained by phage-display technology.

Mechanism-based phage display selection of active-site mutants of human glutathione transferase A1-1 catalyzing SNAr reactions.

A library of active-site mutants has been constructed by targeting selected amino acid residues in human glutathione transferase (GST) A1-1 for random mutagenesis. The mutated residues are suitably positioned for interaction with the second, electrophilic substrate, in particular chloronitrobenzene derivatives undergoing SNAr reactions. DNA representing the GST A1-1 mutant library was fused with DNA encoding gene III protein, a component of the coat of filamentous phage. Phage display was used for affinity selection of GST A1-1 mutants with altered catalytic properties. The affinity ligand used was the sigma-complex of 1,3,5-trinitrobenzene and glutathione immobilized to Sepharose. The complex was designed to mimic the transition state of SNAr reactions catalyzed by GSTs. The selection system is based on the combination of affinity for the sigma-complex as well as the ability to promote its formation, thus mimicking two salient features of the assumed catalytic mechanism for the SNAr reactions. Many of the GST A1-1 mutants selected and analyzed contained an aromatic amino acid residue in one of the mutated positions, suggesting favorable interactions with the trinitrocyclohexadienate moiety of the affinity ligand. A mutant C36 was selected for more detailed studies. Its catalytic efficiency with several chloronitrobenzene substrates was 20-90-fold lower than that of wild-type GST A1-1, but fully comparable to naturally evolved GSTs of different classes, providing a 10(5)-fold rate enhancement over the uncatalyzed reaction. In the conjugation of ethacrynic acid, a Michael addition reaction, mutant C36 was 13-fold more efficient than the wild-type enzyme. Within experimental error, the quotient between the KF values for wild-type GST A1-1 and mutant C36 is the same as that between the kcat/KM values determined with 1-chloro-2,4-dinitrobenzene for the two enzyme forms. This result indicates that sigma-complex formation is rate-limiting for the catalyzed reaction. Thus, the principle of transition-state stabilization as a component of catalysis has been successfully exploited in affinity selection of catalytically competent GST A1-1 mutants. This mechanism-based procedure also selects for the ability to promote sigma-complex formation, and serves as a probe of the catalytic mechanism.

Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1.

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Design of two chimaeric human-rat class alpha glutathione transferases for probing the contribution of C-terminal segments of protein structure to the catalytic properties.

Two chimaeric human-rat class Alpha glutathione transferases were constructed by fusion of DNA segments derived from the plasmids pTGT2-AT and pGTB38 and expression of the corresponding proteins in Escherichia coli. The recombinant proteins H1R1/1 and H1R1/2 encoded by plasmids pH1R1/1 and pH1R1/2 are composed of a segment of the human class Alpha subunit 1 from the N-terminus to His-143 and Pro-207 respectively, followed by the complementary C-terminal portion of the rat class Alpha subunit 1 sequence. Compared with the parental human enzyme, H1R1/1 is altered in 20 positions due to the introduction of 79 residues from the rat enzyme, while H1R1/2 is altered in five positions out of 15 in the C-terminal region. The design of mutant H1R1/1 is equivalent to introduction of exons 6 and 7 of the rat subunit 1 gene in place of the homologous human nucleotide sequence. The two chimaeric proteins are enzymatically active with several substrates, even though the activity in most cases is somewhat decreased in comparison with the wild-type human enzyme. Inhibition studies show that the kinetic properties mimic those of the human enzyme, indicating that the N-terminal two-thirds of the primary structure plays the major role in governing the catalytic properties. The results of this study demonstrate that recombination of segments of primary structure between homologous enzymes may serve as a useful cassette technique for design of novel catalytically active proteins.

Heterologous expression of the allelic variant mu-class glutathione transferases mu and psi.

cDNA encoding the more acidic form, glutathione transferase (GST) psi, of the polymorphic Mu-class GSTs discovered in liver, was mutated in the 5'-end to create an NcoI site, facilitating cloning into the expression plasmid pKK233-2. The protein expressed from this construct has a point mutation Pro-2----Ala-2, but gives a catalytically functional protein. Back-mutation of the codon for amino acid residue 2 gave rise to a plasmid expressing the wild-type enzyme GST psi, or GST Mu1b-1b. A variant cDNA, differing only in specifying lysine rather than asparagine in position 173 of the coding region, was generated by site-directed mutagenesis. The variant sequence corresponds to another cDNA clone isolated from a human liver cDNA library and expresses the near-neutral GST mu, or GST Mu1a-1a. The two recombinant proteins GST Mu1a-1a and GST Mu1b-1b, by physicochemical as well as kinetic criteria, were found to be indistinguishable from GST mu and GST psi respectively, isolated from human liver. It is therefore concluded that the recombinant proteins correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and their protein subunits can be separated by h.p.l.c. on a reverse-phase column. With standard substrates and inhibitors no differences in kinetic parameters between the two variants were detected. The mutated GST Mu1b-1b (Pro-2----Ala) was not significantly different in catalytic properties from the wild-type enzyme, even though Pro-2 is a well conserved amino acid residue in the known Mu-class GSTs.