Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

Wang, R. W.; Newton, Deborah J.; Huskey, Su-Er W.; McKeever, Brian; Pickett, C.B.; Lu, Anthony Y. H.

doi:10.1016/s0021-9258(19)88635-0

Cited by 67 publications

(19 citation statements)

References 47 publications

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“…The pH dependence of k cat /K m CDNB for the reaction with 1-chloro-2,4-dinitrobenzene at saturating concentrations of GSH suggests that the reaction catalyzed by class mu enzymes depends on an ionization with an apparent pK a of about 6.3, a value considerably less than the pK a of 9.0 for GSH in aqueous solution (43,53). Similar results have been reported with the class alpha, pi, and sigma enzymes (31,44,45,54). Spectroscopic observation of the thiolate anion at 240 nm in the active sites of binary complexes of rat isoenzymes M1-1 and M2-2 provides direct evidence that the thiol has a pK a of about 6.7 and that the thiolate anion is the predominant species in the active site at neutral pH, as indicated by the following equation (43,52):…”

Section: E-o-h + Gs-h H E-o-h---supporting

confidence: 73%

“…The theta class enzymes, thought to be the evolutionary precursor of the alpha, mu, pi, and sigma class proteins, appear to utilize the hydroxyl group of a serine residue located near the N-terminus of the polypeptide to activate the sulfhydryl group of bound GSH (32,42). In contrast, the class alpha, mu, pi, and sigma enzymes have recruited the hydroxyl group of a tyrosyl residue, located in a slightly different position, to act as a hydrogen bond donor to the sulfur which lowers the pK a of the thiol in the E‚GSH complex so that it is predominantly ionized at physiological pH (31,(43)(44)(45). Finally, the class alpha enzyme has recruited yet another residue into the inner coordination sphere of the sulfur, the side chain of Arg15, as illustrated in Figure 5 (46).…”

Section: Binding and Activation Of Gshmentioning

confidence: 99%

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Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases

Armstrong

1997

Chem. Res. Toxicol.

1,046

907

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Section: E-o-h + Gs-h H E-o-h---supporting

confidence: 73%

Section: Binding and Activation Of Gshmentioning

confidence: 99%

Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases

Armstrong

1997

Chem. Res. Toxicol.

1,046

907

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“…It is noteworthy that the pK a value of GSH bound to rGST T2-2 was found to be 5.7, similar to that of GSH bound to another theta class GST, hGST T1-1 (33). We believe that this is lower than the pK a s for all other GST-bound GSH described (32,(34)(35)(36)(37)(38).…”

Section: Discussionmentioning

confidence: 62%

“…The unusual pH dependence of k cat , displaying a slow increase with slopes of <1 from pH 4 until the enzyme is finally denatured at pH values above 11 (Figure 4B), can now be explained by the steps in product release (k 6 , k 7 ) determining the steady-state rate of the enzymatic reaction. The pH dependence of such steps would not be expected to be affected by titrations of single groups in the same way as the pH dependences of the catalytic steps of other GSTs studied (32)(33)(34)(35)(36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

Fast Product Formation and Slow Product Release Are Important Features in a Hysteretic Reaction Mechanism of Glutathione Transferase T2-2

Jemth

Mannervik

1999

Biochemistry

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The reaction mechanism of rat glutathione transferase T2-2 has been studied using pre-steady-state and steady-state kinetics. Several parts of the catalytic cycle including binding of substrates, product formation, and product release were investigated. Under saturating conditions, a two-step product release was found to be rate limiting in the enzyme-catalyzed reactions between the nucleophilic substrate glutathione and either of the two electrophilic substrates 1-menaphthyl sulfate and 4-nitrobenzyl chloride. The rate constant for pre-steady-state product formation on rat glutathione transferase T2-2 has an observed pK(a) value of 5.7 apparently due to ionization of the sulfhydryl group of glutathione. This rate constant is approximately 2 orders of magnitude higher than k(cat) at pH values of >6. It can be predicted from the pH dependence that product formation would be the sole rate-limiting step at pH values of <3. A hysteretic mechanism of rGST T2-2 is proposed based on a slow conformational transition detected in pre-steady-state displacement experiments.

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“…One active site feature which is completely conserved in the alpha-, pi-, and mu-class enzymes is a hydrogen bond between the thiol of bound GSH and the phenolic hydroxyl group of a tyrosine side chain. The importance of this hydrogen bond in GST catalysis is clear, based on studies with site-directed mutants (Liu et al, 1992;Wang et al, 1992a;Kong et al, 1992). In addition, spectroscopic experiments have indicated that the pK a of the thiol of GSH is lowered from ∼9.3 in solution to 6.5-6.9 at the active site of GSTs, and that the thiolate anion, GS -, is the predominant speces when bound to the protein at physiological pH (Graminski et al, 1989).…”

mentioning

confidence: 99%

Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

Dietze

Wang

et al. 1996

Biochemistry

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A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.

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Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

Cited by 67 publications

References 47 publications

Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases

Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases

Fast Product Formation and Slow Product Release Are Important Features in a Hysteretic Reaction Mechanism of Glutathione Transferase T2-2

Ligand Effects on the Fluorescence Properties of Tyrosine-9 in Alpha 1-1 Glutathione S-Transferase

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