Toxicity of antirheumatic and anti-inflammatory drugs in children

In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.     

Sulfatases, trapping of the sulfated enzyme intermediate by substituting the active site formylglycine

Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.     

Crystal structure of human arylsulfatase A: the aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis

Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the -CH2SH group of a conserved cysteine to an aldehyde, yielding a formylglycine. Without this modification sulfatases are catalytically inactive, as revealed by a lysosomal storage disorder known as multiple sulfatase deficiency. The 2.1 A resolution X-ray crystal structure shows an ASA homooctamer composed of a tetramer of dimers, (alpha 2)4. The alpha/beta fold of the monomer has significant structural analogy to another hydrolytic enzyme, the alkaline phosphatase, and superposition of these two structures shows that the active centers are located in largely identical positions. The functionally essential formylglycine is located in a positively charged pocket and acts as ligand to an octahedrally coordinated metal ion interpreted as Mg2+. The electron density at the formylglycine suggests the presence of a 2-fold disordered aldehyde group with the possible contribution of an aldehyde hydrate, -CH(OH)2, with gem-hydroxyl groups. In the proposed catalytic mechanism, the aldehyde accepts a water molecule to form a hydrate. One of the two hydroxyl groups hydrolyzes the substrate sulfate ester via a transesterification step, resulting in a covalent intermediate. The second hydroxyl serves to eliminate sulfate under inversion of configuration through C-O cleavage and reformation of the aldehyde. This study provides the structural basis for understanding a novel mechanism of ester hydrolysis and explains the functional importance of the unusually modified amino acid.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus

Acutolysin A alias AaHI, a 22 kDa hemorrhagic toxin isolated from the snake venom of Agkistrodon acutus, is a member of the adamalysin subfamily of the metzincin family and is a snake venom zinc metalloproteinase possessing only one catalytic domain. Acutolysin A was found to have a high-activity and a low-activity under weakly alkaline and acidic conditions, respectively. With the adamalysin II structure as the initial trial-and-error model, the crystal structures were solved to the final crystallographic R-factors of 0. 168 and 0.171, against the diffraction data of crystals grown under pH 5.0 and pH 7.5 conditions to 1.9 A and 1.95 A resolution, respectively. One zinc ion, binding in the active-site, one structural calcium ion and some water molecules were localized in both of the structures. The catalytic zinc ion is coordinated in a tetrahedral manner with one catalytic water molecule anchoring to an intermediate glutamic acid residue (Glu143) and three imidazole Nepsilon2 atoms of His142, His146 and His152 in the highly conserved sequence H142E143XXH146XXGXXH152. There are two new disulfide bridges (Cys157-Cys181 and Cys159-Cys164) in acutolysin A in addition to the highly conserved disulfide bridge Cys117-Cys197. The calcium ion occurs on the molecular surface. The superposition showed that there was no significant conformational changes between the two structures except for a few slight changes of some flexible residue side-chains on the molecular surface, terminal residues and the active-site cleft. The average contact distance between the catalytic water molecule and oxygen atoms of the Glu143 carboxylate group in the weakly alkaline structure was also found to be closer than that in the weakly acidic structure. By comparing the available structural information of the members of the adamalysin subfamily, it seems that, when lowering the pH value, the polarization capability of the Glu143 carboxylate group to the catalytic water molecule become weaker, which might be the structural reason why the snake venom metalloproteinases are inactive or have a low activity under acidic conditions.     

Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.     

Active site labeling of erythrocyte transglutaminase by o-phthalaldehyde

Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.     

Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.     

Distribution of heterochromatin on the mitotic chromosomes of Musca domestica L. in relation to the activity of male-determining factors

Nitric oxide (NO) is a pluripotent regulatory molecule, yet the molecular mechanisms by which it exerts its effects are largely unknown. Few physiologic target molecules of NO have been identified, and even for these, the modifications caused by NO remain uncharacterized. Human glutathione reductase (hGR), a central enzyme of cellular antioxidant defense, is inhibited by S-nitrosoglutathione (GSNO) and by diglutathionyl-dinitroso-iron (DNIC-[GSH]2), two in vivo transport forms of NO. Here, crystal structures of hGR inactivated by GSNO and DNIC-[GSH]2 at 1.7 A resolution provide the first picture of enzyme inactivation by NO-carriers: in GSNO-modified hGR, the active site residue Cys 63 is oxidized to an unusually stable cysteine sulfenic acid (R-SOH), whereas modification with DNIC-[GSH]2 oxidizes Cys 63 to a cysteine sulfinic acid (R-SO2H). Our results illustrate that various forms of NO can mediate distinct chemistry, and that sulfhydryl oxidation must be considered as a major mechanism of NO action.     

Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of beta-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The Km values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.     

Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana)

The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.     

Federated healthcare record server--the Synapses paradigm

Sulfation and sulfate conjugate hydrolysis play an important role in metabolism, and are catalysed by members of the sulfotransferase and sulfatase enzyme super-families. In general, sulfation is a deactivating, detoxication pathway, but for some chemicals the sulfate conjugates are much more reactive than the parent compound. The range of compounds which are sulfated is enormous, yet we still understand relatively little of the function of this pathway. This review summarises current knowledge of the sulfation system and the enzymes involved, and illustrates how heterologous expression of sulfotransferases (SULTs) and sulfatases is aiding our appreciation of the properties of these important proteins. The role of sulfation in the bioactivation of procarcinogens and promutagens is discussed, and new data on the inhibition of the sulfotransferase(s) involved by common dietary components such as tea and coffee are presented. The genetic and environmental factors which are known to influence the activity and expression of human SULTs and sulfatases are also reviewed.     

The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly

We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.     

Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli

The amino acid residues involved in the metal-binding site in the iron-containing dehydrogenase family were characterized by the site-directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron-deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium-chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogenases: (1) V262C produced an enzyme with altered kinetic parameters which nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C-M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resulted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases.     

Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112

The complete amino acid sequence of transglutaminase (EC 2.3.2.13) (TGase), which is produced by a microorganism, Streptoverticillium sp. strain s-8112, and catalyzes the acyl transfer reaction between gamma-carboxyamide groups of glutamine residues in proteins and various primary amines, has been established by a combination of fast atom bombardment mass spectrometry and standard Edman degradation of peptide fragments produced by treatment of the TGase with various proteolytic enzymes and purified by a reversed-phase high performance liquid chromatography. The TGase consists of 331 amino acid residues with a chemical molecular weight of 37,863, in agreement with the observed molecular weight (37,869.2 +/- 8.8) determined from its electrospray ionization mass spectrum. The sequence of the enzyme is very different from those of mammalian TGases represented by guinea pig liver enzyme. The enzyme contains a sole Cys residue, which is essential for its catalytic activity. Hydropathy analysis indicated that the secondary structure of the region around the active site Cys residue is similar to those of mammalian TGases. These results suggest that this microbial protein evolved by a different pathway from that of mammalian TGases and acquired acyl transfer activity during the evolutional process.     

X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase

5-Aminolaevulinate dehydratase (ALAD) is a homo-octameric metallo-enzyme that catalyses the formation of porphobilinogen from 5-aminolaevulinic acid. The structure of the yeast enzyme has been solved to 2.3 A resolution, revealing that each subunit adopts a TIM barrel fold with a 39 residue N-terminal arm. Pairs of monomers wrap their arms around each other to form compact dimers and these associate to form a 422 symmetric octamer. All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263), one of which, Lys 263, forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites one of which is formed by three cysteine residues (133, 135 and 143) while the other involves Cys 234 and His 142. ALAD has features at its active site that are common to both metallo- and Schiff base-aldolases and therefore represents an intriguing combination of both classes of enzyme. Lead ions, which inhibit ALAD potently, replace the zinc bound to the enzyme's unique triple-cysteine site.     

Arylsulfatase from Klebsiella pneumoniae carries a formylglycine generated from a serine

Eukaryotic sulfatases share an unusual posttranslational protein modification, which converts a cysteine into alpha-formylglycine. The alpha-formylglycine is essential for the catalytic activity. Klebsiella pneumoniae expresses an inducible arylsulfatase for which the DNA predicts a serine at the position occupied by the alpha-formylglycine residue in eukaryotic sulfatases. Structural analysis showed that the majority of the arylsulfatase polypeptides from K. pneumoniae carries the alpha-formylglycine, whereas the remaining arylsulfatase polypeptides contain the predicted serine residue. This demonstrates the evolutionary conservation between prokaryotes and eukaryotes of this novel protein modification that so far has been found only in sulfatases. alpha-Formylglycine in Klebsiella is generated from a serine and not from a cysteine as in eukaryotes.