Tyrosine 50 at the Subunit Interface of Dimeric Human Glutathione Transferase P1-1 Is a Structural Key Residue for Modulating Protein Stability and Catalytic Function

2000 ◽  
Vol 271 (1) ◽  
pp. 59-63 ◽  
Author(s):  
Gun Stenberg ◽  
Abdel-Monem Abdalla ◽  
Bengt Mannervik
Keyword(s):  
Protein Stability ◽  
Glutathione Transferase ◽  
Catalytic Function ◽  
Subunit Interface

scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

scholarly journals Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

1990 ◽  
Vol 272 (2) ◽  
pp. 479-484 ◽  
Author(s):  
C Andersson ◽  
R Morgenstern
Keyword(s):  
Chemical Modification ◽  
Rat Liver ◽  
Active Site ◽  
Glutathione Transferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Histidine Residues ◽  
Catalytic Function ◽  
Low Efficiency ◽  
Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

scholarly journals The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability

2005 ◽  
Vol 393 (2) ◽  
pp. 523-528 ◽  
Author(s):  
Carla S. Alves ◽  
Diane C. Kuhnert ◽  
Yasien Sayed ◽  
Heini W. Dirr
Keyword(s):  
Glutathione Transferase ◽  
Tryptophan Fluorescence ◽  
Size Exclusion ◽  
Dimeric Structure ◽  
Subunit Interface ◽  
Exclusion Chromatography ◽  
Region Data ◽  
Glutathione S Transferases ◽  
Urea Unfolding ◽  
Key Residues

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)–HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.

Catalytic Function and Expression of Glutathione Transferase Zeta

2009 ◽  
pp. 85-107 ◽  
Author(s):  
Philip G. Board ◽  
M. W. Anders ◽  
Anneke C. Blackburn
Keyword(s):  
Glutathione Transferase ◽  
Catalytic Function

scholarly journals Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

2003 ◽  
Vol 279 (10) ◽  
pp. 9586-9596 ◽  
Author(s):  
Usama M. Hegazy ◽  
Bengt Mannervik ◽  
Gun Stenberg
Keyword(s):  
Functional Role ◽  
Glutathione Transferase ◽  
Subunit Interface

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1

2010 ◽  
Vol 1804 (12) ◽  
pp. 2228-2233 ◽  
Author(s):  
David Balchin ◽  
Sylvia Fanucchi ◽  
Ikechukwu Achilonu ◽  
Roslin J. Adamson ◽  
Jonathan Burke ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Catalytic Function

Double Mutation at the Subunit Interface of Glutathione Transferase rGSTM1-1 Results in a Stable, Folded Monomer†

Biochemistry ◽  
2006 ◽  
Vol 45 (7) ◽  
pp. 2267-2273 ◽  
Author(s):  
Lawrence C. Thompson ◽  
John Walters ◽  
Jonathan Burke ◽  
James F. Parsons ◽  
Richard N. Armstrong ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Double Mutation ◽  
Subunit Interface

scholarly journals Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination

Symmetry ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 461
Author(s):  
Fereniki Perperopoulou ◽  
Maria Fragoulaki ◽  
Anastassios C. Papageorgiou ◽  
Nikolaos E. Labrou
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Dna Recombination ◽  
Enzyme Reaction ◽  
Optical Biosensor ◽  
Enzyme Design ◽  
Ph Indicator ◽  
Environment Friendly ◽  
Subunit Interface ◽  
Molecular Modeling Studies

In the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class GmGSTUs. The library was activity screened for the identification of glutathione transferase (GST) variants with enhanced catalytic activity towards the herbicide alachlor (2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide). One enzyme variant (GmGSTsf) with improved catalytic activity and binding affinity for alachlor was identified and explored for the development of an optical biosensor for alachlor determination. Kinetics analysis and molecular modeling studies revealed a key mutation (Ile69Val) at the subunit interface (helix α3) that appeared to be responsible for the altered catalytic properties. The enzyme was immobilized directly on polyvinylidenefluoride membrane by crosslinking with glutaraldehyde and was placed on the inner surface of a plastic cuvette. The rate of pH changes observed as a result of the enzyme reaction was followed optometrically using a pH indicator. A calibration curve indicated that the linear concentration range for alachlor was 30–300 μM. The approach used in the present study can provide tools for the generation of novel enzymes for eco-efficient and environment-friendly analytical technologies. In addition, the outcome of this study gives an example for harnessing protein symmetry for enzyme design.

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