Structure Determination and Refinement of Human Alpha Class Glutathione Transferase A1-1, and a Comparison with the Mu and Pi Class Enzymes

Sinning, Irmgard; Kleywegt, Gerard J.; Cowan, Sandra W.; Reinemer, Peter; Dirr, Heini W.; Huber, R.; Gilliland, Gary L.; Armstrong, Richard N.; Ji, Xinhua; Board, Philip G.; Olin, B.; Mannervik, Bengt; Jones, T. Alwyn

doi:10.1006/jmbi.1993.1376

Cited by 432 publications

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“…While this manuscript was in press, the X-ray crystal structure of human Al-1 GST, with S-phenyl GSH bound, was published (Sinning et al, 1993). This structure indicates that Trp 21 lies in a hydrophobic region between domains of the individual subunits.…”

Section: Note Added In Proofmentioning

confidence: 96%

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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Section: Note Added In Proofmentioning

confidence: 96%

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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“…This core contains the loops and adjacent secondary structural elements corre- sponding to motifs 1 and 11, plus the 10 other a-helices and /3-strands, which constitute the GST fold, and which are conserved among GSTM. GSTP, and GSTA (Rushmore C Pickett, 1993; Sinning et al, 1993;Dirr et al, 1994). The result of this threading experiment is thus a set of models based on the GST core and the lowest-energy mapping of the EFl y and GSTT sequences onto this structure (Bryant & Lawrence, 1993).…”

Section: Model Building and Functional Implicationsmentioning

confidence: 99%

“…The subunit is further divided into the N-terminal domain that consists of a 5-stranded 0-sheet and the a-helical C-terminal domain. The GSH-binding site is formed by residues in the N-terminal domain, whereas both the N-terminal and the C-terminal domain contribute to the binding site for the electrophilic GSH acceptor (Ji et al, 1992(Ji et al, , 1994Liu et al, 1992;Reinemer et al, 1992;Rushmore & Pickett, 1993;Sinning et al, 1993;Dirr et al, 1994). The GST-related domain of EFly is predicted to have a similar organization (Fig.…”

Section: Model Building and Functional Implicationsmentioning

confidence: 99%

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

et al. 1994

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Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor ly (EFly) contains an N-terminal domain related to class 0 glutathione S-transferases (GST). GST-like proteins related to class 8 comprise a large group including, in addition to typical GSTs and EFly, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and P-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (a, p, and K). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EFly is likely to form a fold very similar to that in the known structures of class a , p , and ?r GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GSTlike domain in EFly is enzymatically active and that to exhibit GST activity, EFly has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EFl and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.

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“…Analyses of crystal structures of pi, mu, and alpha class glutathione transferases (26 -28) have provided insights into the major interactions between subunits in the homodimers. Their subunit interfaces involve three types of interactions: polar contacts and hydrogen bonds between electrophilic amino acids, stacking of two symmetrically equivalent arginines (one from each subunit) (7,28) and hydrophobic interactions including a lock and key motif. In the lock and key motif an aromatic residue (key residue) from domain I in one subunit is wedged into a hydrophobic pocket formed by helices ␣4 and ␣5 in domain II of the other subunit (Fig.…”

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confidence: 99%

Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

Hegazy¹,

Mannervik

Stenberg

2004

Journal of Biological Chemistry

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The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr 50 in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr 50 to Ala. Although the key residue is located far from the catalytic center, the k cat value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k cat value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr 50 , thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix ␣2, which interacts directly with GSH.

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Structure Determination and Refinement of Human Alpha Class Glutathione Transferase A1-1, and a Comparison with the Mu and Pi Class Enzymes

Cited by 432 publications

References 0 publications

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

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

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

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