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Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions.

Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of kcat/Km, than 3β-hydroxysteroid dehydrogenase/Δ54-isomerase, the enzyme regarded as the mammalian Δ54-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the Δ54-3-ketosteroid isomerase reaction.

GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (kcat/Km) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (kcat) and catalytic efficiency (kcat/Km) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design.

Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead.

The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to kcat and kcat/Km showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , p. 38
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 719
Keywords [en]
Biochemistry
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-2034ISBN: 91-554-5327-9 (print)OAI: oai:DiVA.org:uu-2034DiVA, id: diva2:161674
Public defence
2002-05-28, BMC B22, Uppsala, 10:15
Opponent
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2014-01-27Bibliographically approved
List of papers
1. The role of glutathione in the isomerization of Delta(5)-androstene-3,17-dione catalyzed by human glutathione transferase A1-1
Open this publication in new window or tab >>The role of glutathione in the isomerization of Delta(5)-androstene-3,17-dione catalyzed by human glutathione transferase A1-1
2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 15, p. 11698-11704Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89851 (URN)10.1074/jbc.M009146200 (DOI)11152686 (PubMedID)
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2017-12-14Bibliographically approved
2. Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase
Open this publication in new window or tab >>Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, p. 30019-30022Article in journal (Refereed) Published
Abstract [en]

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89719 (URN)10.1074/jbc.M204485200 (DOI)12023294 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
3. Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
Open this publication in new window or tab >>Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
Show others...
2001 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 51, p. 15835-15845Article in journal (Refereed) Published
Abstract [en]

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89853 (URN)10.1021/bi010429i (DOI)11747461 (PubMedID)
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2017-12-14Bibliographically approved
4. Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism
Open this publication in new window or tab >>Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism
2002 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1598, no 1-2, p. 199-205Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89854 (URN)10.1016/S0167-4838(02)00362-X (DOI)12147362 (PubMedID)
Note

Corrected and republished from Biochim Biophys Acta. 2002 May 20;1597(1):157-63. DOI: 10.1016/S0167-4838(02)00286-8

Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2017-12-14Bibliographically approved

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