| 1. |
- Chaga, Grigoriy, et al.
(författare)
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Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes
- 1994
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 7:9, s. 1115-1119
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Tidskriftsartikel (refereegranskat)abstract
- Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.
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| 2. |
- Danielson, U Helena, et al.
(författare)
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Kinetic independence of the subunits of cytosolic glutathione transferase from the rat
- 1985
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 231:2, s. 263-267
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Tidskriftsartikel (refereegranskat)abstract
- The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.
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| 3. |
- Danielson, U Helena, et al.
(författare)
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Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism
- 1988
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 250:3, s. 705-711
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Tidskriftsartikel (refereegranskat)abstract
- Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.
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| 4. |
- Danielson, U. Helena, et al.
(författare)
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Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site
- 1999
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 38:29, s. 9254-9263
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.
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| 5. |
- Danielson, U Helena, et al.
(författare)
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Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases
- 1987
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 247:3, s. 707-713
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Tidskriftsartikel (refereegranskat)abstract
- The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.
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| 6. |
- Kolm, Rüdiger H., et al.
(författare)
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Isothiocyanates as substrates for human glutathione transferases : structure-activity studies
- 1995
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 311, s. 453-459
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.
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| 7. |
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| 8. |
- Mannervik, Bengt, et al.
(författare)
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Glutathione transferases - structure and catalytic activity
- 1988
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Ingår i: Crc Critical Reviews in Biochemistry. - 0045-6411. ; 23:3, s. 283-337
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Tidskriftsartikel (refereegranskat)abstract
- The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.
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| 9. |
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| 10. |
- Principato, Giovanni B, et al.
(författare)
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Relaxed thiol substrate specificity of glutathione transferase effected by a non-substrate glutathione derivative
- 1988
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 231:1, s. 155-158
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Tidskriftsartikel (refereegranskat)abstract
- Rat glutathione transferase 4-4 catalysed the conjugation of 2-mercaptoethanol with 1-chloro-2,4-dinitrobenzene in the presence of S-methyl-glutathione. The reaction was linearly dependent on enzyme concentration and saturation was seen with respect to both 2-mercaptoethanol and S-methyl-glutathione concentration. High concentrations of S-methyl-gluta-thione were inhibitory. The results suggest that the natural substrate glutathione has two distinct functions in the normal catalytic reaction, (i) induction of a catalytically competent conformation of the enzyme and (ii) provision of the substrate sulfhydryl group in the reaction catalyzed.
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