Polyoxometalates as effective inhibitors for sialyl- and sulfotransferases

Sialylated and/or sulfated carbohydrate chains in glycoproteins and glycolipids play important roles in infection by microorganisms and diseases including cancer. Inhibitors of sialyl/sulfotransferases, responsible for the biosynthesis of these carbohydrate chains, could be medical agents against such infections and diseases. Polyoxometalates (PMs) are inorganic polyanionic molecules that have been shown to exhibit activity against tumors and infectious microorganisms; however, the effects of PMs on carbohydrate biosynthesis have never been investigated. Here, we found that some types of PMs can inhibit the enzymatic activities of specific sialyl/sulfotransferases. Several tungstate-type PMs inhibited Gal: α2,3-sialyltransferase-I (ST3Gal-I) activity at sub-nanomolar levels. The half-inhibitory concentration of the best inhibitors was 0.2 nM and the inhibition was non-competitive for both donor and acceptor substrates (Ki values ∼0.5 nM). By certain vanadate-type PMs, ST3Gal-I and Gal 3-O-sulfotransferase-2 (Gal3ST-2) were specifically inhibited at nanomolar levels. The inhibitory effect of a tungstate-type PM on ST3Gal-I was reversible and electrostatic. A ST3Gal-I mutant protein which was converted ³³⁵Arg residue in the C-terminal region to Glu, was rather insensitive to the PM, suggesting that specific C-terminal basic amino acid of ST3Gal-I is involved in the binding to PMs. Collectively, PMs are novel inhibitors of specific sialyl/sulfotransferases.     

Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms

The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.     

Synthesis and evaluation of conformationally restricted inhibitors of aspartate semialdehyde dehydrogenase

Inhibitors of the enzyme aspartate semialdehyde dehydrogenase, a key biological target for the generation of a new class of antibiotic compounds, have been developed. To investigate improvements to binding within an inhibitor series, the lowering of the entropic barrier to binding through conformational restriction was investigated. A library of linear and cyclic substrate analogues was generated and computational docking used to aid in structure selection. The cyclic phosphonate inhibitor 18 was thus identified as complimentary to the enzyme active-site. Synthesis and in vitro inhibition assay revealed a K(i) of 3.8 mM against natural substrate, where the linear analogue of 18, compound 15, had previously shown no inhibitory activity. Two further inhibitors, phosphate analogue diastereoisomers 17a and 17b, were synthesised and also found to have low millimolar K(i) values. As a result of the computational docking investigations, a novel substrate binding interaction was discovered: hydrogen bonding between the substrate (phosphate hydroxy-group as the hydrogen bond donor) and the NADPH cofactor (2'-oxygen as the hydrogen bond acceptor).     

Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function

Galectins are β-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely β-galactoside α2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an α2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to β-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.     

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.     

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.     

Genetically altered mice with different sialyltransferase deficiencies show tissue-specific alterations in sialylation and sialic acid 9-O-acetylation

Glycan chains on glycoconjugates traversing the Golgi apparatus are often terminated by sialic acid residues, which can also be 9-O-acetylated. This process involves competition between multiple Golgi enzymes. Expression levels of Golgi enzyme mRNAs do not always correlate with enzyme activity, which in turn cannot accurately predict glycan sequences found on cell surfaces. Here we examine the cell type-specific expression of terminal glycans in tissues of normal mice in comparison with animals deficient in ST6Gal-I (transfers alpha2-6-linked sialic acid to Galbeta1-4GlcNAc) or ST3Gal-I (transfers alpha2-3-linked sialic acid to Galbeta1-3GalNAc). Tissues of ST6Gal-I null mice showed minimal binding of an alpha2-6-sialic acid-specific lectin, indicating that no other enzyme generates Siaalpha2-6Galbeta1-4GlcNAc and that Siaalpha2-6GalNAc (sialyl-Tn) is rare in mice. However, exposed Galbeta1-4GlcNAc termini were only moderately increased, indicating that these can be partially capped by other enzymes. Indeed, Galalpha1-3Galbeta1-4GlcNAc and Fucalpha1-2Galbeta1-4GlcNAc termini were enhanced in some tissues. Many tissues of ST3Gal-I null animals showed increases in Galbeta1-3GalNAc termini, and some increases in poly-N-acetyllactosamines. However, overall expression of alpha2-3-linked sialic acid was selectively reduced only in a few instances, indicating that other ST3Gal enzymes can generate this linkage in most tissues. Highly selective losses of 9-O-acetylation of sialic acid residues were also observed, with ST6Gal-I deficiency causing loss on endothelium and ST3Gal-I deficiency giving a marked decrease on CD4(+) lymphocytes. These data demonstrate selective regulation of sialylation and 9-O-acetylation, point to cell types with potential physiological defects in null animals, and show in vivo evidence for competition between Golgi enzymes.     

The sialyltransferase ST3Gal-I is not required for regulation of CD8-class I MHC binding during T cell development

The CD8 coreceptor plays a crucial role in thymocyte and T cell sensitivity by binding to class I MHC and recruiting downstream signaling molecules to the TCR. Previous studies reported considerable changes in TCR-independent CD8/class I MHC binding (i.e., CD8 noncognate interactions) during T cell development, changes that correlated with altered glycosylation of surface molecules. In particular, expression of the sialyltransferase ST3Gal-I has been proposed as a critical factor regulating the attenuation of CD8 avidity during the double-positive to CD8 single-positive progression. This hypothesis is strengthened by the fact that ST3Gal-I(-/-) animals show a profound disregulation of CD8 T cell homeostasis. In contrast to this model, however, we report in this study that ST3Gal-I deficiency had no detectable impact on CD8 noncognate binding to multimeric peptide/MHC class I ligands at any stage of thymocyte development. We also found that the susceptibility to CD8-induced cell death is not markedly influenced by ST3Gal-I deficiency. Thus, the profound effects of ST3Gal-I on CD8 T cell survival evidently do not involve a role for this enzyme in controlling CD8-class I binding.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells

Tumor-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of carcinoma cells. The aim of this study was to examine the effect of alpha 2,6-sialylation on the adhesion properties of breast carcinoma cells. To this end mammary carcinoma cells, MDA-MB-435, were sense-transfected with sialyltransferase ST6Gal-I cDNA or antisense-transfected with a part of the ST6Gal-I sequence. Sense transfectants showed an enhanced ST6Gal-I mRNA expression and enzyme activity and an increased binding of the lectin Sambucus nigra agglutinin (SNA), specific for alpha 2,6-linked sialic acid. Transfection with ST6Gal-I in the antisense direction resulted in less enzyme activity and SNA reactivity. A sense-transfected clone carrying increased amounts of alpha 2,6-linked sialic acid adhered preferentially to collagen IV and showed reduced cell-cell adhesion and enhanced invasion capacity. In contrast, antisense transfection led to less collagen IV adhesion but enhanced homotypic cell-cell adhesion. In another approach, inhibition of ST6Gal-I enzyme activity by application of soluble antisense-oligodeoxynucleotides was studied. Antisense treatment resulted in reduced ST6 mRNA expression and cell surface 2,6-sialylation and significantly decreased collagen IV adhesion. Our results suggest that cell surface alpha 2,6-sialylation contributes to cell-cell and cell-extracellular matrix adhesion of tumor cells. Inhibition of sialytransferase ST6Gal-I by antisense-oligodeoxynucleotides might be a way to reduce the metastatic capacity of carcinoma cells.     

Proteolytic shedding of ST6Gal-I by BACE1 regulates the glycosylation and function of alpha4beta1 integrins

Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of alpha4beta1 integrins. Here we report that the sustained alpha4beta1 activation associated with macrophage differentiation results from expression of beta1 integrin subunits that lack alpha2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14(+) monocytes, ST6Gal-I is down-regulated, leading to beta1 hyposialylation and enhanced alpha4beta1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, beta1 hyposialylation, and alpha4beta1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the beta1 subunit. Collectively, these results describe a novel mechanism for alpha4beta1 regulation and further suggest an unanticipated role for BACE1 in macrophage function.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.     

Structure-based inhibitor design for an enzyme that binds different steroids: a potent inhibitor for human type 5 17beta-hydroxysteroid dehydrogenase

Human type 5 17beta-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30 A. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9+/-1.4 nM, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy.     

Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase

The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 A. The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit. Both active sites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible absorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme. The propionate-bound structure therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one of the residues interacting with the carboxylate group of propionate, Arg136. We propose that this novel interaction influences both substrate binding and catalysis by precisely positioning Arg136 and modulating its charge.     

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.