Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (kcat/Km) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti- and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest kcat/Km value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionylation. In conclusion, the present study indicates that individuals who are homozygous for the allele GSTP1* B (coding for GSTP1-1/V-105) display a higher susceptibility to malignancy because of other factors than a decreased catalytic efficiency of GSTP1-1/V-105 in the detoxication of carcinogenic diol epoxides of benzo[a]pyrene or structurally related PAH.

Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.

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Human class Alpha glutathione transferase (GST) Al-l has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Ty$+Phe lowers the specific activities with three substrates to 2-8% of the values for the wi!d-type enzyme. The changes in the kinetic parameters &;ca,iKu. I',.,, and S,, show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered k,,, values. suggesting that the hydroxyl group of Tyr* is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (199 1) EMBO J. 10. 1997-20051. Thus. Tyr" appears to be the first active site residue established as participating in the chemical mechanism of a GST.Glutathione transferase; Human: Site-directed mutagenesis: Active-site tyrosine 1, INTRODUCTION 2. MATERIALS AND METHODSComparison c,f primary structures of homologous proteins is one approach that can be used to establish amino acid residues of significance for the structure and function of protein molecules. In the cytosolic glutathione transferases characterized. about 5% of the approximately 220 amino acid residues are conserved in all the major classes of the enzymes [l]. Some of these are arginine residues and a recent investigation based on site-directed mutagenesis indicated that such residues are possibly involved in binding of the cofactor glutathione [2]. A tyrosine residue near the N-terminus is also highly conserved [I], and has been targeted for mutagenesis experiments. In the work described here this residue (Tyr') has been mutated into Phe in human class Alpha glutathione transferase Al-l*. The relevance of this mutation became accentuated when an X-ray diffraction investigation demonstrated that the corresponding tyrosine residue in a pig lung class Pi enzyme is positioned close to a glutathione analogue in the active site [3]. The results of the present investigation strongly suggest that Tyr' contributes to enzyme catalysis. even though it is not essential for catalysis to occur. AI-1 has also been ~~amrd GST2 type I. GST, and GSTB,B, I. CiwtriicdsSephadex G-25 and epoxy-activated Scpharose 6B were from Pharmacia LKB Biotechnology. Uppsala, Sweden. S-Hexylglutathione and S-hexylglutathione Sepharose 6B were synthesized as previously described [4], Substrates for enzyme activity measurements and other chemicals were of highest quality available from commercial sources. d'-Androstene-3.1 'I-dione was generously provided by Dr. Paul Talalay (Johns Hopkins University, Baltimore. MD. USA). Oligonuclcotide-directed in vitro mutagenesis kit and [?'P]dCTP were from Amersham Intcmational. Restriction enzymes. and other DNA-modifying enzymes as well as plasmids were obtained from Amersham International (Promega Corp., Boehringer Mannheim and Pharmacia LKB Biotechnology). Mutagenic oligonucleotides were synthesized at the Department of Immunology. Uppsala University, ...