Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2‐2

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k cat and k cat / K m(GSH) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.

Section: Resultssupporting

confidence: 52%

“…GSH, S-hexylglutathione, CDNB, and 1-fluoro-2,4-dinitrobenzene (FDNB) were Sigma products. His-tagged recombinant hGSTT2-2 and R107A mutant were expressed in Escherichia coli and purified using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as described previously (13,17).…”

Section: Methodsmentioning

confidence: 99%

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

Caccuri

Antonini

Board

et al. 2001

“…GSTT 2-2 acts as a sulfatase with menaphthyl sulfate and generates menaphthyl glutathione and free sulfate. Mutagenesis has suggested that this reaction is not dependent on the presence of a serine and may be a product of the environment generated by a number of residues (43,44). This notable difference demonstrates that some reactions catalyzed by GSTs can take place in the absence of a tyrosine or serine residue, and an equivalent mechanism may be utilized by the Omega class GSTs.…”

Section: Discussionmentioning

confidence: 99%

Identification, Characterization, and Crystal Structure of the Omega Class Glutathione Transferases

Board¹,

Coggan²,

Chelvanayagam³

et al. 2000

658

647

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-Å resolution and has a characteristic GST fold (Protein Data Bank entry code 1eem). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

“…Expression of the human GSTT2-2 and its purification using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen, Hilden, Germany) were carried out as reported previously (35,36). Expression and purification of the bacterial GSTB1-1 was performed as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

The Specific Interaction of Dinitrosyl-Diglutathionyl-Iron Complex, a Natural NO Carrier, with the Glutathione Transferase Superfamily

Maria

Pedersen

Caccuri

et al. 2003

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (K D Х 10 ؊10 M), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (K D Х 10 ؊9 M). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (K D Х 10 ؊7 M), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.