Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl ␣-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl ␣-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pK a of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.

Section: Enzymementioning

confidence: 99%

Section: Enzymementioning

confidence: 99%

An Electron-sharing Network Involved in the Catalytic Mechanism Is Functionally Conserved in Different Glutathione Transferase Classes

Winayanuwattikun¹,

Ketterman

2005

“…Recent years have witnessed a tremendous proliferation of three-dimensional structure determinations by the advent of high throughput x-ray crystallography soon after the sequence and adequate expression of a protein became available (1,2). In some proteins, no electron density can be observed for stretches of the amino acid sequence especially at the N or C terminus (3)(4)(5)(6)(7)(8). Thus, a functional role for such regions is not always discernible from their three-dimensional structures.…”

mentioning

confidence: 99%

“…Thus, a functional role for such regions is not always discernible from their three-dimensional structures. The catalytic domain of botulinum neurotoxin (BoNT) 3 belongs to this category of proteins. BoNT and tetanus neurotoxins are a unique class of zinc endopeptidases that act selectively at discrete sites on three synaptosomal proteins of the neuroexocytotic apparatus (for reviews, see Refs.…”

mentioning

confidence: 99%

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Rahman

Frasca²,

Swaminathan

et al. 2013

Background:The function of C terminus of botulinum neurotoxin catalytic domain is unknown. Results: Synthetic C-terminal peptides competitively inhibited but at stoichiometric concentrations stimulated serotype A proteolytic activity. Conclusion: C terminus interacts with the active site and may function by removing a product. Significance: The inhibition and product removal appear to be a unique feature of type A botulinum neurotoxin among catalytic proteins.

“…2 The class Alpha GSTs possess, like many other GST classes, an active site tyrosine residue for the activation of GSH but, unlike other GST classes, they possess an extended C-terminal region that forms an integral part of the active site (5-7). The major structural element in the C-terminal region is the amphipathic helix 9 that, although not directly involved in the chemical mechanism of catalysis, is an important determinant of substrate selectivity (8 -11), the binding of substrates (12), rate-determining steps (13), desolvation of the active site (14), and the pK a of the catalytic tyrosine residue (12,15). In addition to its contributions to catalysis, the C-terminal region is an important determinant of ligandin function, i.e.…”

mentioning

confidence: 99%

A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases

Dirr

Little

Kuhnert

et al. 2005

Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.