Renal cell carcinoma risk in type 2 von Hippel-Lindau disease correlates with defects in pVHL stability and HIF-1alpha interactions.

The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate binding subunit of the CBC(VHL) E3 ubiquitin ligase complex. Mutations in the VHL gene cause a variety of tumors with complex genotype/phenotype correlations. Type 2A and type 2B VHL disease are characterized by a low or high risk of renal cell carcinoma, respectively. To investigate the molecular basis underlying the difference between disease types 2A and 2B, we performed a detailed biochemical analysis of the two most frequent type 2A mutations, Y98 H and Y112 H, in comparison to type 2B mutations in the same residues, Y98N and Y112N. While none of these mutations affected the assembly of CBC(VHL) complexes, the type 2A mutant proteins exhibited higher stabilities at physiological temperature. Moreover, the type 2A mutant proteins possessed higher binding affinities for the key cellular substrate, hypoxia-inducible transcription factor 1 (HIF-1alpha). Consistent with these results, type 2A but not type 2B mutant VHL proteins retained significant ubiquitin ligase activity towards HIF-1alpha in vitro. We propose that this residual ubiquitin ligase activity is sufficient to suppress renal cell carcinogenesis in vivo.

Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.

Measurement of glutathione transferases.

There are multiple glutathione transferase genes, the proteins for which have different substrate specificities. The various genes are differentially expressed such that species and organs and tissues differ qualitatively and quantitatively for cytosolic and membrane-bound forms. This unit provides protocols for analysis of transferase activity in a continuous spectrophotometric assay and an assay with dichloromethane as the substrate.

Yeast glyoxalase I is a monomeric enzyme with two active sites.

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.

The active-site residue tyr-175 in human glyoxalase II contributes to binding of glutathione derivatives.

Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to catalysis. The mutation had a marginal effect on the k(cat) value determined using S-D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K(m) value than the wild-type enzyme. The competitive inhibitor S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione had a 30-fold higher K(i) value towards the mutant, than that of the wild-type. Pre-equilibrium fluorescence studies with the inhibitor showed that this was due to a significantly increased off-rate for the mutant enzyme. The phenolic hydroxyl group of tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that this interaction makes a significant contribution to the binding of the active-site ligand.

Active site serine promotes stabilization of the reactive glutathione thiolate in rat glutathione transferase T2-2. Evidence against proposed sulfatase activity of the corresponding human enzyme.

Ser(11) in rat glutathione transferase T2-2 is important for stabilization of the reactive enzyme-bound glutathione thiolate in the reaction with 1-menaphthyl sulfate. The S11A mutation increased the pK(a) value for the pH dependence of the rate constant for pre-steady-state product formation, from 5.7 to 7.9. This pH dependence is proposed to reflect titration of enzyme-bound glutathione thiol. Further, the mutation lowered the k(cat) value but not because of the impaired stabilization of the glutathione thiolate. In fact, several steps on the reaction pathway were affected by the S11A mutation, and the cause of the decreased k(cat) for the mutant was found to be a slower product release. The data presented here contradict the hypothesis that glutathione transferase T2-2 could act as a sulfatase that is not dependent on Ser(11) for the catalytic activity, as proposed for the corresponding human enzyme (Tan, K.-L., Chelvanayagam, G., Parker, M. W., and Board, P. G. (1996) Biochem. J. 319, 315-321; Rossjohn, J., McKinstry, W. J., Oakley, A. J., Verger, D., Flanagan, J., Chelvanayagam, G., Tan, K.-L., Board, P. G., and Parker, M. W. (1998) Structure 6, 309-322). On the contrary, Ser(11) governs both chemical and physical steps of the catalyzed reaction.

The C-terminal region of human glutathione transferase A1-1 affects the rate of glutathione binding and the ionization of the active-site Tyr9.

In human glutathione transferase (GST) A1-1, the C-terminal region covers the active site and contributes to substrate binding. This region is flexible, but upon binding of an active-site ligand, it is stabilized as an amphipatic alpha-helix. The stabilization has implications for the catalytic activity of the enzyme. In the present study, residue M208 in GST A1-1 has been mutated to Lys and Glu, and residue F220 to Ala and Thr. These mutations are likely to destabilize the C-terminal region due to loss of hydrophobic interactions with the rest of the hydrophobic binding site. The rate constant for binding of glutathione to wild-type GST A1-1 is 450 mM(-)(1) s(-)(1) at 5 degrees C and pH 7.0, which is less than for an association limited by diffusion. However, the M208 and the F220 mutations increase the apparent on-rate constant for glutathione binding to 640-1170 mM(-)(1) s(-)(1). The binding data can be explained by a rapid reversible transition between different enzyme conformations occurring prior to glutathione binding, and restriction of the access to the active site by the C-terminal region. The effect of the mutations appears to be promotion of a less closed conformation, thereby facilitating the association of glutathione and enzyme. Both the M208 and F220 mutants display a lowered pK(a) value ( approximately 0.3 log unit) of the catalytically important Tyr9. Residue 208 does not interact directly with Tyr9 in the active site, and the shift in pK(a) value is therefore ascribed to the proposed dislocation of the C-terminal region caused by the mutation.

Fast product formation and slow product release are important features in a hysteretic reaction mechanism of glutathione transferase T2-2.

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.

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Phospholipid hydroperoxide glutathione peroxidase activity of human glutathione transferases.

Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order: GST A1-1>GST T1-1>GST M1-1>GST A2-2>GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide,1-palmitoyl-2-(13-hydroper oxy-cis-9, trans-11 -octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol/min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-dependent phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx), the presence of detergent inhibited the activity of GST A1-1 on PLPC-OOH. Also, in contrast with Se-PHGPx, only glutathione could act as the reducing agent for GST A1-1. A GST A1-1 mutant (Arg15Lys), which retains the positive charge between the GSH- and hydrophobic binding sites, exhibited a decreased kcat for PLPC-OOH but not for CDNB, suggesting that the correct topography of the GSH site is more critical for the phospholipid substrate. A Met208Ala mutation, which gives a modified hydrophobic site, decreased the kcat for CDNB and PLPC-OOH by comparable amounts. These results indicate that Alpha, Mu and Theta class human GSTs provide protection against accumulation of cellular phospholipid hydroperoxides.

Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases.

Thymine hydroperoxide (5-hydroperoxymethyluracil), a model compound representing products of oxidative damage to DNA, is a substrate for glutathione peroxidase and some isoforms of glutathione transferase. In this paper, we show that selenium-dependent human phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx) exhibits about four orders of magnitude higher activity on thymine hydroperoxide than that of other human enzymes such as selenium-dependent glutathione peroxidase and various representatives of glutathione transferases. The results indicate that Se-PHGPx may be an important enzyme in repairing oxidatively damaged DNA.

Kinetic characterization of recombinant human glutathione transferase T1-1, a polymorphic detoxication enzyme.

Recombinant human theta class glutathione transferase T1-1 has been heterologously expressed in Escherichia coli and a simple purification method involving immobilized ferric ion affinity chromatography and Orange A dye chromatography is described. The catalytic properties of the enzyme differ significantly from those of other glutathione transferases, also within the theta class, with respect to both substrate selectivity and kinetic parameters. In addition to 1,2-epoxy-3-(4-nitrophenoxy)propane, the substrate used previously to monitor the enzyme, human glutathione transferase T1-1 has activity with the naturally occurring phenethylisothiocyanate and also displays glutathione peroxidase activity with cumene hydroperoxide. Further, the enzyme is active with 4-nitrobenzyl chloride and 4-nitrophenethyl bromide, but shows no detectable activity with the more chemically reactive 1-chloro-2,4-dinitrobenzene. The Michaelis constant for glutathione, K(m)GSH, with 1,2-epoxy-3-(4-nitrophenoxy)propane as second substrate, is high at low pH values but decreases at higher pH values. This is mirrored in kcat/K(m)GSH which increases with an apparent pKa value of 9.0, reflecting the ionization of the thiol group of glutathione in solution. The same results are obtained with 4-nitrophenethyl bromide as electrophilic substrate, although the K(m)GSH value (0.72 mM at pH 7.5), as well as the pKa (8.1) derived from the pH dependence of kcat/K(m)GSH, are lower with this substrate. In contrast, kcat and kcat/K(m)electrophile display either a maximum or a plateau at pH 7.0-7.5, and an apparent pKa value of 5.7 was determined for the pH dependence of kcat with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates. This pKa value reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pKa value thus is lowered by the enzyme. Three differences in the cDNA as compared to the sequence previously published were found. One of these differences causes a change in the deduced amino acid sequence and involves the nucleotide triplet encoding amino acid 126, which was determined as GAG (Glu), instead of the published GGG (Gly).

Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10).