An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.     

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

The present study characterized conserved residues in a GST (glutathione transferase) in the active-site region that interacts with glutathione. This region of the active site is near the glycine moiety of glutathione and consists of a hydrogen bond network. In the GSTD (Delta class GST) studied, adGSTD4-4, the network consisted of His(38), Met(39), Asn(47), Gln(49), His(50) and Cys(51). In addition to contributing to glutathione binding, this region also had major effects on enzyme catalysis, as shown by changes in kinetic parameters and substrate-specific activity. The results also suggest that the electron distribution of this network plays a role in stabilization of the ionized thiol of glutathione as well as impacting on the catalytic rate-limiting step. This area constitutes a second glutathione active-site network involved in glutathione ionization distinct from a network previously observed interacting with the glutamyl end of glutathione. This second network also appears to be functionally conserved in GSTs. In the present study, His(50) is the key basic residue stabilized by this network, as shown by up to a 300-fold decrease in k(cat) and 5200-fold decrease in k(cat)/K(m) for glutathione. Although these network residues have a minor role in structural integrity, the replaced residues induced changes in active-site topography as well as generating positive co-operativity towards glutathione. Moreover, this network at the glycine moiety of GSH (glutathione) also contributed to the 'base-assisted deprotonation model' for GSH ionization. Taken together, the results indicate a critical role for the functionally conserved basic residue His(50) and this hydrogen bond network in the active site.     

Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium

Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS(-); in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a pi-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pK(a) of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison.     

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.     

Contribution of glycine 146 to a conserved folding module affecting stability and refolding of human glutathione transferase p1-1

In human glutathione transferase P1-1 (hGSTP1-1) position 146 is occupied by a glycine residue, which is located in a bend of a long loop that together with the alpha6-helix forms a substructure (GST motif II) maintained in all soluble GSTs. In the present study G146A and G146V mutants were generated by site-directed mutagenesis in order to investigate the function played by this conserved residue in folding and stability of hGSTP1-1. Crystallographic analysis of the G146V variant, expressed at the permissive temperature of 25 degrees C, indicates that the mutation causes a substantial change of the backbone conformation because of steric hindrance. Stability measurements indicate that this mutant is inactivated at a temperature as low as 32 degrees C. The structure of the G146A mutant is identical to that of the wild type with the mutated residue having main-chain bond angles in a high energy region of the Ramachandran plot. However even this Gly --> Ala substitution inactivates the enzyme at 37 degrees C. Thermodynamic analysis of all variants confirms, together with previous findings, the critical role played by GST motif II for overall protein stability. Analysis of reactivation in vitro indicates that any mutation of Gly-146 alters the folding pathway by favoring aggregation at 37 degrees C. It is hypothesized that the GST motif II is involved in the nucleation mechanism of the protein and that the substitution of Gly-146 alters this transient substructure. Gly-146 is part of the buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), conserved in all GSTs and related proteins that seems to behave as a characteristic structural module important for protein folding and stability.     

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k_cat regulation. Surprisingly, the T11A mutant had a decreased GSH K_m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.     

Glutathione-Binding Site of a Bombyx mori Theta-Class Glutathione Transferase

The glutathione transferase (GST) superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT) catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.     

Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue

Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase.

Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.     

Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases

The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.     

Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages

It has been previously reported that the three-dimensional structures of the NAD-binding and catalytic site of bacterial toxins with ADP-ribosylating activity are superimposable, and that the key amino acids for the enzymatic activity are conserved. The model includes an NAD-binding and catalytic site formed by an α-helix bent over a β-strand, surrounded by two β-strands bearing a Glu and a His, or Arg, that are required for catalysis. We show here that the model can be extended to comprise all proteins with ADP-ribosylating activity known to date, including all eukaryotic mono- and poly-ADP-ribosyltransferases, the bacterial ADP-ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T-even bacteriophages. We show that, in addition to the common Glu and Arg/His amino acids previously identified, the conserved motifs can be extended as follows: (i) the Arg/His motif is usually arom-His/Arg (where 'arom' is an aromatic residue); (ii) in the sequences of the CT group the β-strand forming part of the 'scaffold' of the catalytic cavity has an arom-ph-Ser-Thr-Ser-ph consensus (where 'ph' represents a hydrophobic residue); and (iii) the motif centred in the key glutamic residue is Glu/Gln-X-Glu; while (iv) in the sequences of the DT group the NAD-binding motif is Tyr-X₁₀-Tyr. We believe that the model proposed not only accounts for all ADP-ribosylating proteins known to date, but it is likely to fit other enzymes (currently being analysed) which possess such an activity.     

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.     

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.     

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1

The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly.     

Identification of two essential glutamic acid residues in glycogen synthase

The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not known. Through the multiple alignment of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the members of this family. This peptide includes an E-X(7)-E motif, which is highly conserved in four families of retaining glycosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (Glu-510 and Glu-518) of the motif. Proteins were transiently expressed in COS-1 cells as fusions to green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in response to glucose and to bind to intracellular glycogen. Although the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion protein, the E510A mutation inactivated the enzyme. These results led us to conclude that the E-X(7)-E motif is part of the active site of eukaryotic glycogen synthases and that both conserved Glu residues are involved in catalysis. We propose that Glu-510 may function as the nucleophile and Glu-518 as the general acid/base catalyst.