Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1
| Autor: | Ikechukwu Achilonu, Jonathan Burke, Roslin J. Adamson, Manuel A. Fernandes, Samantha Gildenhuys, Sylvia Fanucchi, Heini W. Dirr, David Balchin |
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| Rok vydání: | 2010 |
| Předmět: |
Models
Molecular Protein Denaturation Stereochemistry Protein subunit Biophysics Crystallography X-Ray Biochemistry Analytical Chemistry Substrate Specificity Transition state analog Catalytic Domain Enzyme Stability Dinitrochlorobenzene Transferase Humans Amino Acid Sequence Molecular Biology Glutathione Transferase Alanine Binding Sites biology Sequence Homology Amino Acid Chemistry Circular Dichroism Temperature Tryptophan Substrate (chemistry) Active site Glutathione Meisenheimer complex Protein Structure Tertiary Isoenzymes Kinetics Spectrometry Fluorescence Helix Mutation biology.protein Biocatalysis Protein Binding |
| Zdroj: | Biochimica et biophysica acta. 1804(12) |
| ISSN: | 0006-3002 |
| Popis: | Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the SNAr reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain–domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs. |
| Databáze: | OpenAIRE |
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