The reactive serine residue of epidermolytic toxin A.

Comparison of amino acid sequence data suggested that there may be a functional relationship between the staphylococcal epidermolytic toxins and V8 proteinase. The hypothesis was tested by treating epidermolytic toxin with di-isopropyl phosphorofluoridate, which bound specifically at serine-195, the homologue of the active-site serine residue of V8 proteinase.     

Mutational analysis of the superantigen staphylococcal exfoliative toxin A (ETA)

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.     

The epidermolytic toxins are serine proteases

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF-PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin-like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca2(+)-binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium-binding loop of rat intestinal calcium-binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca2(+)-dependent mechanism.     

Effect of anesthetics on the stability of oxyhemoglobin S

The conversion of the serine-195 in α-chymotrypsin to dehydroalanine results in two conformational substates that differ in their extinction coefficients at 240nm. The active site methionine-192 in the substate with lower absorption at 240nm is alkylated by α-bromo-4-nitroacetophenone at a rate of 7.0×10^?4sec^?1, similar to that found for α-chymotrypsin; the substate with higher absorption at 240nm reacts 14 times slower. These two substates are not separated by an affinity resin containing lima bean trypsin inhibitor. These data infer that the serine-195 plays a role in the stabilization of the active site conformation in α-chymotrypsin.     

Inhibition of in-vitro fertilization of hamster oocytes by a thiol proteinase inhibitor

Amongst the proteinase inhibitors tested, thiolstatin, a specific inhibitor for the thiol proteinases, leupeptin and antipain, both specific inhibitors of serine- and thiol-proteinases, strongly reduced fertilization of hamster oocytes in vitro. These results suggest the possible involvement of thiol proteinase(s), as well as acrosin, in the fertilization process. A possible role for thiol proteinase in sperm adhesion to the zona pellucida is proposed.     

Multiplex PCR for detection of three exfoliative toxin serotype genes inStaphylococcus aureus

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.     

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate the same site on HMG-CoA reductase as the AMP-activated protein kinase

Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate and inactivate both intact, microsomal HMG-CoA reductase, and the purified 53 kDa catalytic fragment. Isolation of the single phosphopeptide produced by combined cleavage with cyanogen bromide and Lys-C proteinase reveals that this is due to phosphorylation of a single serine residue near the C-terminus, corresponding to serine-872 in the human enzyme. This is identical with the single serine phosphorylated by the AMP-activated protein kinase. The nature of the protein kinase responsible for phosphorylation of this site in vivo is discussed.     

Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553.

A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) carrying a deletion of glutamic acid-553, an important active-site residue, was expressed in an ETA-negative strain of P. aeruginosa and shown to be exported from the cells as efficiently as wild-type ETA. The mutant protein, purified from the culture medium, was devoid of ADP-ribosyltransferase activity. Protein conformation was barely perturbed by the deletion, as determined by a number of measures, including affinity for substrate NAD, proteinase sensitivity, absorbance and fluorescence spectroscopy, and differential scanning calorimetry. The conformational integrity and stability of the mutant toxin are consistent with potential use of the protein in vaccines or as a carrier in preparing conjugate vaccines.     

Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism.

The use of a linear free-energy relationship shows that both histidine residues of alpha-chymotrypsin and chymotrypsinogen are super-reactive toward 1-fluoro-2,4-dinitrobenzene. The binding of indole to the specificity site of alpha-chymotrypsin causes both histidine residues to become less reactive. On the basis of these results and those from X-ray-crystallographic studies, the following conclusions are made. (1) The super-reactivity of the catalytic-site histidine-57 is due to charge transfer from aspartic acid-102 by means of hydrogen bonding. (2) The aspartic acid-102-histidine-57-serine-195 'charge-relay' system is not complete in the zymogen or native enzyme and only on binding of a suitable substrate or ligand to the specificity site of the enzyme is the charge transfer to serine-195 completed. (3) The lack of substantial enzymic activity in the zymogen is due to the absence of a completed specificity site, and therefore it cannot bind suitable substrates or ligands to induce completion of the charge-relay system.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

A metalloproteinase associated with gelonin, a "ribosome inactivating protein"

Gelonin, a single-chain protein which inactivates eukaryotic ribosomes, becomes split into peptides when incubated with SDS. During the chromatographic purification of gelonin on carboxymethylcellulose three overlapping peaks emerge in the gelonin elution region, containing three proteins with small differences in apparent molecular weight (31,500, 30,000 and 29,200). All three proteins are endowed with inhibitory activity on protein synthesis and with proteinase activity, although with different specific activities, and all three give rise to the same peptides upon incubation with SDS, suggesting that they are isoforms of gelonin. The gelonin-associated proteinase acts only on gelonin, while it is inactive on the most common substrates for endoproteinases. The proteolytic activity is not inhibited by inhibitors of serine- or SH-proteinases, while it is completely abolished by chelating agents. Divalent cations restore the proteolytic activity inhibited by EDTA. The stability of the proteinase activity on exposure of gelonin to extreme values of pH or to prolonged incubation has been investigated. The inhibitory activity on protein synthesis and the proteinase activity are differently affected by these treatments.     

Primary structure of streptococcal proteinase. III. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain.

The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.     

Complete amino acid sequence of the collagenase from the insect Hypoderma lineatum

The primary structure of the Hypoderma lineatum collagenase was determined. Chymotrypsin digestion and thermolysin fragmentation of the chymotryptic core gave 30 and 5 peptides, respectively, accounting for all the residues of the protein. These peptides were aligned with overlapping peptides derived from tryptic and Staphylococcus aureus V8 proteinase digests. Hypoderma collagenase is a serine proteinase composed of 230 amino acids (Mr 25,223). It displays a high degree of sequential homology with the serine proteinases of the trypsin family, especially with another collagenolytic enzyme, the proteinase I of the crab Uca pugilator. The six half-cystinyl residues of Hypoderma collagenase correspond to 6 of the 10 half-cystinyl residues of chymotrypsin, and the residues forming the charge-relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions. The prediction of the secondary structure of the collagenase is given.     

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.     

X-ray diffraction analysis of the inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone

The inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. A covalent adduct is formed that deacylates slowly with a half-life of 23 h. X-ray diffraction analysis at 1.9-A resolution of the inactivator-enzyme complex shows that the gamma-oxygen of the active-site serine (serine-195) is covalently attached to C-1 of (Z)-2-benzylpentenedioic acid, the benzyl group of the inactivator is held in the hydrophobic specificity pocket of the enzyme, and the free carboxylate forms a salt bridge with the active-site histidine (histidine-57). The conformational changes that occur in the protein as a result of complexation are described. It is proposed that formation of the salt bridge prevents access of water and, therefore, hydrolysis of the acyl-enzyme.     

Nucleotide sequence of the epidermolytic toxin A gene of Staphylococcus aureus.

The nucleotide sequence of the eta gene, which codes for the epidermolytic toxin serotype A of Staphylococcus aureus TC16, is reported. The coding sequence of 840 nucleotides specifies a protein which, when secreted, has a predicted molecular weight of 26,950. The sequence of eta and the deduced amino acid sequence of the toxin have been compared with those of epidermolytic toxin serotype B. The coding sequences have 52% identical residues, and the polypeptides have 40% identical residues. Amino acid residues have been conserved in the areas of the proteins which correspond to major hydrophobic domains, whereas the regions likely to specify antigenic determinants occur in hydrophilic sequences that have diverged. The level of expression of epidermolytic toxin A in S. aureus 8325-4 was shown to be dependent on the integrity of a regulatory gene called agr.     

Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy

The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombin(S195A)) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys(232), Cys(P3'), and Cys(313)) were covalently modified with the fluorescence probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change upon forming a complex with either active or inactive proteinase. Steady-state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombin(S195A) is also able to induce a conformational change in the serpin.