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1.
Johan Ålander Johan Lengqvist Peter J. Holm Pascal Gerbaux Hans Hebert Richard N. Armstrong 《Archives of biochemistry and biophysics》2009,487(1):42-856
The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (Kd = 320 ± 50 μM). All three product molecules could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, Kd could be determined for a low affinity GSH-binding site (2.5 ± 0.5 mM). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent. 相似文献
2.
Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues. 相似文献
3.
Johan Ålander Vanina Dahlström Heuser Julia Järvliden Aya Shibata Yoshihiro Ito 《Analytical biochemistry》2009,390(1):52-1708
A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were kcat = 0.075 ± 0.005 s−1 and Km = 21 ± 3 μM (kcat/Km = 3.6 × 103 ± 5.6 × 102 M−1 s−1), giving a rate enhancement of 106 compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes. 相似文献
4.
Fedulova N Raffalli-Mathieu F Mannervik B 《Archives of biochemistry and biophysics》2011,507(2):205-211
An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1∗. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence. 相似文献
5.
Siritantikorn A Johansson K Ahlen K Rinaldi R Suthiphongchai T Wilairat P Morgenstern R 《Biochemical and biophysical research communications》2007,355(2):592-596
Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis. 相似文献
6.
We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties. 相似文献
7.
Philip G. Board Yang Yu Xiao Han Jingyan Wei 《Journal of molecular recognition : JMR》2013,26(1):38-45
Accumulating evidence shows that glutathione peroxidase (GPX, EC.1.11.1.9), one of the most important antioxidant selenoenzymes, plays an essential role in protecting cells and tissues against oxidative damage by catalyzing the reduction of hydrogen peroxide by glutathione. Unfortunately, because of the limited availability and poor stability of GPX, it has not been used clinically to protect against oxidative stress. To overcome these problems, it is necessary to generate mimics of GPX. In this study, we have used directed mutagenesis and the inclusion of a selenocysteine (Sec) insertion sequence to engineer the expression in eukaryotic cells of human glutathione transferase zeta1–1 (hGSTZ1–1) with Sec in the active site (seleno‐hGSTZ1–1). This modification converted hGSTZ1–1 into an active GPX and is the first time this has been achieved in eukaryotic cells. The GPX activity of seleno‐hGSTZ1–1 is higher than that of GPX from bovine liver, indicating Sec at the active site plays an important role in the determination of catalytic specificity and performance. Kinetic studies revealed that the ping–pong catalytic mechanism of Se‐hGSTZ1–1 is similar to that of the natural GPX. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
8.
Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1. 相似文献
9.
Chonticha Saisawang Jantana Wongsantichon Robert C. Robinson Albert J. Ketterman 《Proteins》2019,87(7):588-595
In the human neuroblastoma SH-SY5Y cell line, the glutathione transferase Omega 1-1 (GSTO1-1) appears to modulate Akt and MEK1/2 kinase activation. We observed a glutathionylation modification was involved in the activation of Akt but not MEK1/2. With the specific GSTO1-1 inhibitor ML175, we show the enzyme activity of GSTO1-1 is important for modulation as the inhibited GSTO1-1 allowed activation of both Akt and MEK1/2. The inhibition of GSTO1-1 showed a similar extent of activation of Akt and MEK1/2 as treatment by the endotoxin lipopolysaccharide. The GSTO1-1 also either directly interacts with Akt and MEK1/2 or interacts with a protein complexed with Akt and MEK1/2 as both kinases coimmunoprecipitated with GSTO1-1. The results suggest that GSTO1-1 enzyme activity inhibits the activation of these two kinases to maintain basal levels. The possible regulation by GSTO1-1 is of interest as both kinases have hundreds of potential downstream targets that are known to have contributions to various cellular processes including survival, growth, proliferation, and metabolism. 相似文献
10.
Shield AJ Murray TP Board PG 《Biochemical and biophysical research communications》2006,347(4):859-866
Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found. 相似文献
11.
A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. 相似文献
12.
R Fabrini E Rosato A Gigante A Bocedi R Cianci B Barbano E Del Grosso F Ricci V Zingaretti F Salsano G Ricci 《Cell death & disease》2013,4(7):e736
Erythrocyte glutathione transferase (e-GST) is a detoxifying enzyme hyper-expressed in nephropathic patients and used recently as a biomarker for blood toxicity. Systemic sclerosis (SSc) is characterized by endothelial dysfunction and fibrosis of the skin and internal organs. Renal involvement is frequent in SSc patients. Here we show that e-GST is hyper-expressed in SSc patients (n=102) and correlates (R2=0.49, P<0.0001) with the Medsger DSS and DAI Valentini indices that quantify the severity and activity of this disease. Interestingly, e-GST does not correlate with the impairment of kidney or other specific organs taken separately. e-GST hyper-expression seems to be linked to the presence of a factor (i.e., toxin) that triggers the autoimmune disease, and not to the damage of specific organs or to oxidative stress. e-GST may be proposed as an innovative non-antibody biomarker for SSc useful to check the progress of this disease and the efficiency of new therapeutic strategies. 相似文献
13.
Tars K Larsson AK Shokeer A Olin B Mannervik B Kleywegt GJ 《Journal of molecular biology》2006,355(1):96-105
The crystal structures of wild-type human theta class glutathione-S-transferase (GST) T1-1 and its W234R mutant, where Trp234 was replaced by Arg, were solved both in the presence and absence of S-hexyl-glutathione. The W234R mutant was of interest due to its previously observed enhanced catalytic activity compared to the wild-type enzyme. GST T1-1 from rat and mouse naturally contain Arg in position 234, with correspondingly high catalytic efficiency. The overall structure of GST T1-1 is similar to that of GST T2-2, as expected from their 53% sequence identity at the protein level. Wild-type GST T1-1 has the side-chain of Trp234 occupying a significant portion of the active site. This bulky residue prevents efficient binding of both glutathione and hydrophobic substrates through steric hindrance. The wild-type GST T1-1 crystal structure, obtained from co-crystallization experiments with glutathione and its derivatives, showed no electron density for the glutathione ligand. However, the structure of GST T1-1 mutant W234R showed clear electron density for S-hexyl-glutathione after co-crystallization. In contrast to Trp234 in the wild-type structure, the side-chain of Arg234 in the mutant does not occupy any part of the substrate-binding site. Instead, Arg234 is pointing in a different direction and, in addition, interacts with the carboxylate group of glutathione. These findings explain our earlier observation that the W234R mutant has a markedly improved catalytic activity with most substrates tested to date compared to the wild-type enzyme. GST T1-1 catalyzes detoxication reactions as well as reactions that result in toxic products, and our findings therefore suggest that humans have gained an evolutionary advantage by a partially disabled active site. 相似文献
14.
Rinaldi R Aniya Y Svensson R Eliasson E Swedmark S Shimoji M Morgenstern R 《Chemico-biological interactions》2004,147(2):163-172
Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted. 相似文献
15.
Micaloni C Kong GK Mazzetti AP Nuccetelli M Antonini G Stella L McKinstry WJ Polekhina G Rossjohn J Federici G Ricci G Parker MW Lo Bello M 《Journal of molecular biology》2003,325(1):111-122
We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme. 相似文献
16.
Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity 总被引:8,自引:0,他引:8
SunHZ WuSF 《Cell research》2001,11(2):107-115
INTRODUCTIONtransitional cell carcinoma (TCC) of the bladder represents the fifth most preValent malignancy inwestern population. A major problem in the management of TCC is the low sensitivity to chemotherapy and the high recu-rrence after transurethral resection, which occupies a large proportion (approximately 40%) among bladder cancer patients[1, 21. Sodrug resistance remains a major and difficult problem to resolye in TCC chemotherapy. This phenomenon has often been ascribed to so… 相似文献
17.
The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme. 相似文献
18.
Kapoli P Axarli IA Platis D Fragoulaki M Paine M Hemingway J Vontas J Labrou NE 《Biosensors & bioelectronics》2008,24(3):498-503
Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20muM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme. 相似文献
19.
Muhammet Serhat Özaslan Yeliz Demir Hatice Esra Aslan Şükrü Beydemir Ömer İrfan Küfrevioğlu 《Journal of biochemical and molecular toxicology》2018,32(5)
Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play an important role in cellular signaling. In the present study, potential inhibition effects of chalcones were tested against human GST. For this purpose, GST was purified from human erythrocytes with 5.381 EU?mg?1 specific activity and 51.95% yield using a GSH–agarose affinity chromatographic method. The effects of chalcones on in vitro GST activity were tested at various concentrations. Ki constants of chalcones were found in the range of 7.76–41.93 μM. According to the results, 4‐fluorochalcone showed a better inhibitory effect compared with the other compounds. The inhibition mechanisms of 2'‐hydroxy‐4‐methoxychalcone and 4‐methoxychalcone were noncompetitive, whereas the inhibition mechanisms of 4'‐ hydroxychalcone, 4‐ fluorochalcone, and 4,4'‐ diflurochalcone were competitive. 相似文献
20.
Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities. 相似文献