Structure of the phosphosaccharide-inositol core of the Leishmania donovani lipophosphoglycan

The phosphosaccharide-inositol core of the lipophosphoglycan of Leishmania donovani was generated by treatment of the glycoconjugate with mild acid and digestion with phosphatidylinositol-specific phospholipase C. The core was purified and examined by one- and two-dimensional 1H-1H NMR and by methylation analysis. From the results, the carbohydrate core was elucidated as a phosphosaccharide attached to the inositol residue of the lyso-alkylphosphatidylinositol anchor of lipophosphoglycan as follows: PO4----6GalP(alpha 1----6)GalP(alpha 1----3)Galf(alpha 1----3)ManP(alpha 1----3)ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol. The presence of an internal galactofuranose residue is highly unusual and the ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol sequence is homologous to the respective portion of the glycosylphosphatidylinositol anchors reported for both the Trypanosoma brucei variant surface glycoprotein and the rat brain Thy-1 glycoprotein.     

Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.     

Assessment of the sequence dependency for the binding of 2-aminonaphthyridine to the guanine bulge

We have studied the sequence dependent binding of 2-amino-1,8-naphthyridine derivative 1 to a single guanine bulge. The free energy changes for the binding to a guanine bulge with different sequence contexts (5'X_Y3'/3'X'GY'5') were determined by a curve fitting of the thermal denaturation profile of DNA in the presence and absence of 1. The data showed that (i) the binding of 1 to a guanine bulge is stronger for those flanking the G-C base pair than A-T base pair, (ii) the guanine 3' side to 1 in the complex is especially effective for the complex stabilization, and (iii) the increase of T(m) in the presence of 1 is not a good estimate for the sequence dependent binding. The most efficient 1-binding was observed for the sequence of G_G/CGC. Molecular modeling simulations suggested that stacking interaction between the 3' side guanine and 1 is the molecular basis for the strong binding to G_G/CGC.     

Determination of the relative contributions of the diselenide and selenol forms of ebselen in the mechanism of its glutathione peroxidase-like activity.

The molecular basis of the glutathione peroxidase activity of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) was investigated by the use of synthesised, authentic intermediates identical to those formed by the reaction of ebselen with glutathione. The second order rate constants for the reaction of ebselen (0.29 mM-1 min-1), ebselen-glutathione selenosulfide (less than or equal to 0.01 mM-1 min-1), ebselen selenol (2.8 mM-1 min-1) and ebselen diselenide (0.32 mM-1 min-1) with hydrogen peroxide reveal that the selenol is particularly active in this respect. The determination of the relative amounts of ebselen selenol and diselenide under typical peroxidase assay conditions implies that the selenol is the predominant molecular species responsible for the glutathione--(70%)--and dithiothreitol--(96%)--dependent peroxidase activity of ebselen.     

Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.     

Characterization of the nucleotide tight-binding sites of the isolated mitochondrial F1-ATPase

The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.     

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the alpha1beta1 and the alpha1beta2 interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the alpha1beta1 and alpha1beta2 interfaces as reflected by 1H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment alpha(28-35) is in a helical conformation, while the segment alpha(19-22) is the nonhelical AB region. The shortened B-helix conserves the interactions of alpha1beta1 interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of alpha(23-26) without perturbing its overall global conformation.     

The effect of metal ions on the electrochemistry of the antitumor antibiotic streptonigrin

The effect of transition metal ions on the electrochemistry of 6-methoxy-5,8-quinolinedione (L1), 7-amino-6-methoxy-5,8-quinolinedione (L2) and the antitumor antibiotic streptonigrin (SN) was studied. In 10% methanol/water, the one-electron reduction of quinones L1 and L2 to the corresponding semiquinones is shifted to more positive potentials upon addition of one equivalent of Zn(II), Ni(II), Co(II) or Cd(II) and is consistent with formation of a 1:1 complex involving the quinone(N) and adjacent quinone(O). Similar results are observed for Cu(II) and Mn(II), but the redox chemistry is also complicated by metal-based redox chemistry. The addition of further equivalents of M(II) results in a number of different coordination and electrochemical processes including formation of 1:1 and 2:1 complexes of the quinone, semiquinone and dianion. Under similar conditions, the 1:1 SN 2,2'-bipyridyl metal complex undergoes a reversible one-electron reduction to the semiquinone. The redox potential of the quinone in SN was shifted positive in the presence of the metal ions, but both the magnitude of the shift, and the relative influence of the metals was different to ligands L1 and L2. The changes in redox chemistry of SN compared with L1 and L2 are consistent with the formation of the 2,2-bipyridyl complexes in which there is weaker coordination to the quinone(O) in ring A of SN. These results suggest that in vivo, metal ions such as Zn(II), Cu(II) and Mn(II) facilitate the initial reduction of streptonigrin to the semiquinone by capturing the semiquinone after SN is reduced by biological reductants.     

Changes at the interface of the N- and C-terminal parts of the heavy chain of myosin subfragment 1

It has been previously shown that in the M-MgADP-P(i) state, where the myosin head adopts a pre-power stroke conformation, treatment of trypsin-split subfragment 1 of skeletal muscle myosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) results in cross-linking of the C-terminal fragment of the heavy chain of S1 -- most probably its converter region -- to the N-terminal S1 heavy-chain fragment, generating a product of 44 kDa [Biochim. Biophys. Acta 1481 (2000) 55]. The results described here show that this product is neither generated in the absence of nucleotide nor in the presence of MgADP or MgPP(i). The 44 kDa cross-linking product can be formed when S1 treated with EDC is complexed with MgADP-AlF(4) or MgADP-V(i) (MgADP-P(i) analogs) and with MgADP-BeF(x), MgATP gamma S or MgAMPPNP (MgATP analogs). The results suggest structural differences between MgATP- or MgADP-P(i)-bound S1, and MgADP-bound or nucleotide-free S1, in spatially close regions of their N- and C-terminal heavy-chain fragments.     

Thermosynthesis as energy source for the RNA World: a model for the bioenergetics of the origin of life

The thermosynthesis concept, biological free energy gain from thermal cycling, is combined with the concept of the RNA World. The resulting overall origin of life model suggests new explanations for the emergence of the genetic code and the ribosome. It is proposed that the first protein named pF(1) obtained the energy to support the RNA World by a thermal variation of F(1) ATP synthase's binding change mechanism. It is further proposed that this pF(1) was the single translation product during the emergence of the genetic machinery. During thermal cycling pF(1) condensed many substrates with broad specificity, yielding NTPs and randomly constituted protein and RNA libraries that contained self-replicating RNA. The smallness of pF(1) permitted the emergence of the genetic machinery by selection of RNA that increased the fraction of pF(1)s in the protein library: (1) an amino acids concatenating progenitor of rRNA bound to (2) a chain of 'positional tRNAs' linked by mutual recognition, and yielded a pF(1) (or its main motif); this positional tRNA set gradually evolved to a set of regular tRNAs functioning according to the genetic code, with concomitant emergence of (3) an mRNA coding for pF(1).     

Participation of the C-terminal region of the D1-polypeptide in the first steps in the assembly of the Mn4Ca cluster of photosystem II

Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.     

Molecular biology of the CRH receptors-- in the mood

Dysfunctioning of corticotropin-releasing hormone (CRH) and its receptors (CRH(1) and CRH(2)) has been linked to the development of stress-related disorders, such as mood and eating disorders. The molecular characterization of CRH(1) and CRH(2) receptors and their splice variants has generated detailed information on their pharmacology, tissue distribution and physiology. While mammalian CRH(1) receptors nonselectively bind CRH analogs, the ligand specificity of CRH(2) is narrower. CRH(1) receptors are predominantly expressed in the brain and pituitary, whereas CRH(2) receptor expression is limited to particular brain areas and to some peripheral organs. Molecular approaches to block CRH(1) receptor expression in the brain argue in favor of its involvement in the regulation of some aspects of the stress response. The CRH(2alpha) receptor may be more important for motivational types of behavior essential for survival, such as feeding and defense.(1)     

Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1

The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in [Ca(2+)](i) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca(2+)](i) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca(2+)](i) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca(2+)](i) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.     

Head-head/tail-tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP

BACKGROUND: The transporter associated with antigen processing (TAP) is a heterodimeric member of the large family of ABC transporters. The study of interactions between the subunits TAP1 and TAP2 can reveal the relative orientation of the transmembrane segments, which form a translocation pore for peptides. This is essential for understanding the architecture of TAP and other ABC transporters. RESULTS: The amino-terminal six transmembrane segments (TMs) of human TAP1, TAP1 (1-6), and the amino-terminal five TMs of TAP2, TAP2(1-5), are thought to constitute the pore of TAP. Two new approaches are used to define dimer interactions. We show that TM6 of TAP1 (1-6) is able to change topology post-translationally. This TM, along with a cytoplasmic tail, is translocated into the endoplasmic reticulum lumen, unless TAP2 is expressed. Coexpression of TM(4-5) of TAP2 stabilizes the topology of TAP1 (1-6), even when the TM1 of TAP1 is subsitituted with another sequence. This suggests that the carboxy-terminal TMs of the pore-forming domains TAP1 (1-6) and TAP2(1-5) interact. An alternative assay uses photobleaching in living cells using TAP1 (1-6) tagged with the green fluorescent protein (GFP). Coexpression with TAP2(1-5) results in reduced movement of the heterodimer within the endoplasmic reticulum membrane, as compared with the single TAP1 (1-6) molecule. In contrast, TAP2(1-4) has no effect on the mobility of TAP1 (1-6)-GFP, indicating the importance of TM5 of TAP2 for dimer formation. Also, TM1 of both TAP1 and TAP2 is essential for formation of a complex with low mobility. CONCLUSIONS: Dimerization of the pore-forming transmembrane domains of TAP1 (TM1-6) with its TAP2 counterpart (TM1-5) prevents the post-translational translocation of TM6 of TAP1 and results in a complex with reduced mobility within the endoplasmic reticulum membrane compared with the free subunit. These techniques are used to show that the pore-forming domains of TAP are aligned in a head-head/tail-tail orientation. This positions the following peptide-binding segments of the two TAP subunits to one side of the pore.     

Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide

The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r. techniques being the main methods used. It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose. ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.     

On the kinetics of the reaction between human antiplasmin and a low-molecular-weight form of plasmin.

The reaction between antiplasmin (A) and a low-molecular-weight form of plasmin (P) proceeds in at least two steps: a fast reversible second-order reaction followed by a slower irreversible first-order transition, and may be represented by: P +A k1 in equilibrium k-1 PA k2 leads to PA'. The low-Mr plasmin, which is obtained by limited elastase digestion, is composed of an intact B chain and a small A chain lacking the lysine-binding sites. The k1 of the reaction is (6.5 +/- 0.5) x 10(5) M-1 s-1 which is 30--60 times smaller than that for normal plasmin and antiplasmin. The dissociation constant of the first step is 1.9 x 10(-9) M which is 10 times higher than for normal plasmin and antiplasmin. The rate constant of the second step is (4.2 +/- 0.2) x 10(-3) s-1 for both normal and low-Mr plasmin. Low Mr plasmin which has substrate bound to its active site does not react or reacts only very slowly with antiplasmin. The reaction rate, however, is only slightly influenced by 6-aminohexanoic acid in concentrations up to 1 mM which decrease the reaction rate of normal plasmin approximately 50-fold. The findings further indicate that the lysine-binding site(s) of plasmin are of great importance for the rate of its reaction with antiplasmin.     

Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii

The lipopolysaccharide of Bordetella hinzii was analyzed after various chemical degradations by NMR spectroscopy and MALDI mass spectrometry, and the following structure of the polysaccharide chain was determined: 4-O-Me-alpha-GalpNAc3NAcAN-(1-->[-->4)-beta-GlcpNAc3NAcAN-(1-->4)-beta-GlcpNAc3NAcAN-(1-->4)-alpha-GalpNAc3NAcAN-(1-](n)-where GlcNAc3NAcAN and GalNAc3NAcAN stand for 2,3-diacetamido-2,3-dideoxy-glucuronamide and -galacturonamide, respectively. The polysaccharide chain is terminated with a 4-O-methylated GalNAc3NAcAN residue and is rather short (n < or = 5).     

Structure of the lipophosphoglycan from Leishmania major

The major cell surface glycoconjugate of the parasitic protozoan Leishmania major is a heterogeneous lipophosphoglycan. It has a tripartite structure, consisting of a phosphoglycan (Mr 5,000-40,000), a variably phosphorylated hexasaccharide glycan core, and a lysoalkylphosphatidylinositol (lysoalkyl-PI) lipid anchor. The structures of the phosphoglycan and the hexasaccharide core were determined by monosaccharide analysis, methylation analysis, fast atom bombardment-mass spectrometry, one- and two-dimensional 500-MHz (correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA] 1H NMR spectroscopy, and exoglycosidase digestions. The phosphoglycan consists of eight types of phosphorylated oligosaccharide repeats which have the general structure, [formula: see text] where R = H, Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3), Glcp(beta 1-3)Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3), or Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), and where all the monosaccharides, including arabinose, are in the D-configuration. The average number of repeat units/molecule (n) is 27. Data are presented which suggest that the nonreducing terminus of the phosphoglycan is capped exclusively with the neutral disaccharide Manp(alpha 1-2)Manp alpha 1-. The structure of the glycan core was determined to be, [formula: see text] where approximately 60% of the mannose residues distal to the glucosamine are phosphorylated and where the inositol is part of the lysoalkyl-PI lipid moiety containing predominantly 24:0 and 26:0 alkyl chains. The unusual galactofuranose residue is in the beta-configuration, correcting a previous report where this residue was identified as alpha Galf. Although most of the phosphorylated repeat units are attached to the terminal galactose 6-phosphate of the core to form a linear lipophosphoglycan (LPG) molecule, some of the mannose 6-phosphate residues may also be substituted to form a Y-shaped molecule. The L. major LPG is more complex than the previously characterized LPG from Leishmania donovani, although both LPGs have the same repeating backbone structure and glycolipid anchor. Finally we show that the LPG anchor is structurally related to the major glycolipid species of L. major, indicating that some of these glycolipids may have a function as precursors to LPG.     

Total synthesis of the natural antigen involved in the hyperacute rejection response to xenotransplants

The major glycosphingolipid in pig vascular endothelium is the ceramide pentasaccharide Gal alpha(1 --> 3)Gal beta(1 --> 4)GlcNAc beta(1 --> 3)Gal beta(1 --> 4)Glc beta(1 --> 0)Cer (1), which binds specifically to human anti-Gal antibody and is involved in the hyperacute rejection response in xenotransplantation from pig to man. The synthesis of 1 and its methyl glycoside 2 is described.     

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.