Site-directed mutagenesis of glutathione S-transferase YaYa: Nonessential role of histidine in catalysis

Wang, Regina W.; Newton, Deborah J.; Pickett, Cecil B.; Lu, Anthony Y. H.

doi:10.1016/0003-9861(91)90082-t

Cited by 39 publications

(24 citation statements)

References 24 publications

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“…9). At equilibrium, three variations are possible for the position of the hydrogen bond between the active site Tyr and the thiol group of gluta- The catalytic role of Tyr has been investigated by sitedirected mutagenesis in which the Tyr was replaced with Phe [58][59][60][61][62][63][64]1271, His, Thr, or Val [63]. These substitutions are not tolerated and result in mutants with severely impaired catalytic activities but with near wild-type-like glutathione binding affinities.…”

Section: An Evolutionarily Conserved G-site Tyrosine and The Activatimentioning

confidence: 99%

“…These substitutions are not tolerated and result in mutants with severely impaired catalytic activities but with near wild-type-like glutathione binding affinities. In two of the mutants, the hydrogen-bond donor moieties in the side chains of His and Thr are not close enough to the sulphur atom of glutathione and are therefore unable to interact with and activate the tripeptide's thiol group [63]. The Tyr+Phe mutation does not significantly affect the stereoselective behaviour of class mu-catalyzed addition reactions with a,p-unsaturated ketones [61].…”

Section: An Evolutionarily Conserved G-site Tyrosine and The Activatimentioning

confidence: 99%

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X‐ray crystal structures of cytosolic glutathione S‐transferases

Dirr

Reinemer

Huber

1994

European Journal of Biochemistry

416

321

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Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pK,) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases. Portrait of a superfamily of detoxification enzymesMany sophisticated defense strategies have evolved in organisms enabling them to deal with the constant threat by a broad spectrum of both foreign and endogenous cytotoxic and genotoxic compounds [1]. Enzyme systems that transact the chemical detoxification and elimination processes of these structurally diverse molecules, many of which are highly non-polar, can be divided into three major but interrelated groups; phase I enzymes, such as the cytochrome P450 superfamily, activate chemicals by forming reactive functional groups (e.g. epoxides) in them; phase I1 enzymes deactivate these reactive chemicals by appending a hydrophilic moiety (e.g. glutathionyl, glucuronyl, or sulphuryl) to the functional group ; phase Ill enzymes mediate the cellular elimination of the inactive and water-soluble products.The superfamily of glutathione S-transferases (EC 2.5.1.18) represents an integral part of the phase I1 detoxification mechanism. These intracellular proteins are found in most aerobic eukaryotes and prokaryotes, and protect cells against chemical-induced toxicity and stress by catalyzing the S-conjugation between the thiol group of glutathione and an electrophilic moiety in the hydrophobic and toxic sub- GST, glutathione S-transferase; pGSTP1-1, hGSTAl-1, rGSTM1-1 etc., acronyms for the glutathione S-transferases (GST), the prefix indicating the species (p, porcine; h, human ; r, rat; b, bovine; m, mouse; rb, rabbit; c, chicken; gp, guinea pig) while P, A, and M indicate gene class pi, alpha and mu, respectively ; 1-1 indicates a dimer of two type-1 subunits; GSH, reduced glutathione; P1, P2 etc. and G1, G2 etc., designate peptide functional groups in glutathione and the corresponding G-site ligands of the glutathione S-transferases, respectively; G-site, glutatbione-binding site; H-site, hydrophobic electrophile-binding site.Enzyme. Glutathione S-transferase (EC 2.5.1.1 8).strate. Glutathione S-transferases are perhaps the single most important family of enzymes involved in the metabolism of alkylating compounds [2]. The resultant glutathione S-conjugates can be exported from animal cells by putative membrane ATP-dependent pump systems [3] after which they are metabolized via the mercapturate pathway and eventually eliminated. An extraordinary feature of the glutathione S-transferase superfamily is the occurrence of multiple enzyme forms that, according to sequence similarities and subc...

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Section: An Evolutionarily Conserved G-site Tyrosine and The Activatimentioning

confidence: 99%

Section: An Evolutionarily Conserved G-site Tyrosine and The Activatimentioning

confidence: 99%

X‐ray crystal structures of cytosolic glutathione S‐transferases

Dirr

Reinemer

Huber

1994

European Journal of Biochemistry

416

321

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“…The histidine residues of rat GST 1-1 (His9, His143 and His159), a member of the a family and has 58% identity with CL 3-3, have been mutated by Wang et al (1991). His143 and His159 on GST 1-1 are the equivalent of His142 and His158 on CL 3-3.…”

Section: Enzyme Activitymentioning

confidence: 99%

Site‐directed mutagenesis and chemical modification of histidine residues on an α‐class chick liver glutathione S‐transferase CL 3‐3

Chang¹,

Tam²

1993

European Journal of Biochemistry

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Each chick liver glutathione S-transferase CL 3 subunit contains three histidine residues : His142, His158 and His228. CL 3-3 can be inactivated by treating with diethylpyrocarbonate. The inactivation process is pH dependent and the pK, of the modified residue is 6.4. The second-order inhibition rate constant is 741 M-Imin-' at pH 7.0. Based on difference-spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethylpyrocarbonate-modified enzyme only partially restored the activity (30-50%) of CL 3-3. By tryptic mapping and amino acid sequence analysis, His228 and Lysl4 have been identified as the modified residues.Mutants with histidine to serine replacement (H142S and H158S) or C-terminal histidine deletion (des-H228) were constructed and over-expressed in Spodopteru frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des-H228 mutant can be inactivated by diethylpyrocarbonate. These results support the conclusion that histidines are not involved in the enzymic mechanism of CL 3-3.

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“…The N-terminal domain (domain I) contains both P-sheet and a-helical elements, whereas the larger C-terminal domain (domain 11) contains no &sheet structure. The glutathione-binding site ("G-site") lies nearly completely within the N-terminal domain of each subunit, yet includes a single salt bridge between GSH bound to one subunit and an evolutionarily conserved aspartate in the adjacent subunit (Wang et al, 1992a;Kong et al, 1993). Notably, the GSH-binding site lies near the N-termini of a-helices 1 and 3, and it has been suggested that the resulting helical dipoles may play an electrostatic role in GSH binding (Graminski et al, 1989;Reinemer et al, 1991).…”

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Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

et al. 1993

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The glutathione S-transferase (GST) isoenzyme Al-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within a-helix 1 based on comparison with the X-ray crystal structures of the pi-and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme Al-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequencydomain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substratefree GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16070 of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

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Site-directed mutagenesis of glutathione S-transferase YaYa: Nonessential role of histidine in catalysis

Cited by 39 publications

References 24 publications

X‐ray crystal structures of cytosolic glutathione S‐transferases

X‐ray crystal structures of cytosolic glutathione S‐transferases

Site‐directed mutagenesis and chemical modification of histidine residues on an α‐class chick liver glutathione S‐transferase CL 3‐3

Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

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