Characterization of Active-Site Residues of the NIa Protease from Tobacco Vein Mottling Virus

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased k_cat marginally by about 4.7-fold and increased K_m by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in k_cat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pK_m values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.     

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.     

Structure of a ternary complex of proteinase K,mercury, and a substrate-analogue hexa-peptide at 2.2 Å resolution

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH₂ has been determined at 2.2 Å resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 Sγ which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide. © 1996 Wiley-Liss, Inc.     

Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus mouse hepatitis virus gene 1 is expressed as a polyprotein, which is cleaved into multiple proteins posttranslationally. One of the proteins is p28, which represents the amino-terminal portion of the polyprotein and is presumably generated by the activity of an autoproteinase domain of the polyprotein (S. C. Baker, C. K. Shieh, L. H. Soe, M.-F. Chang, D. M. Vannier, and M. M. C. Lai, J. Virol. 63:3693-3699, 1989). In this study, the boundaries and the critical amino acid residues of this putative proteinase domain were characterized by deletion analysis and site-directed mutagenesis. Proteinase activity was monitored by examining the generation of p28 during in vitro translation in rabbit reticulocyte lysates. Deletion analysis defined the proteinase domain to be within the sequences encoded from the 3.6- to 4.4-kb region from the 5' end of the genome. A 0.7-kb region between the substrate (p28) and proteinase domain could be deleted without affecting the proteolytic cleavage. However, a larger deletion (1.6 kb) resulted in the loss of proteinase activity, suggesting the importance of spacing sequences between proteinase and substrate. Computer-assisted analysis of the amino acid sequence of the proteinase domain identified potential catalytic cysteine and histidine residues in a stretch of sequence distantly related to papain-like cysteine proteinases. The role of these putative catalytic residues in the proteinase activity was studied by site-specific mutagenesis. Mutations of Cys-1137 or His-1288 led to a complete loss of proteinase activity, implicating these residues as essential for the catalytic activity. In contrast, most mutations of His-1317 or Cys-1172 had no or only minor effects on proteinase activity. This study establishes that mouse hepatitis virus gene 1 encodes a proteinase domain, in the region from 3.6 to 4.4 kb from the 5' end of the genome, which resembles members of the papain family of cysteine proteinases and that this proteinase domain is responsible for the cleavage of the N-terminal peptide.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.     

Studies on the active sites ofBacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Summary Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites ofBacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced inBacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G towardp-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP andp-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities.     

Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease.

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis.     

Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo.

The NIa protein of plant potyviruses is a bifunctional protein containing an N-terminal VPg domain and a C-terminal proteinase region. The majority of tobacco etch potyvirus (TEV) NIa molecules are localized to the nucleus of infected cells, although a proportion of NIa is attached covalently as VPg to viral RNA in the cytoplasm. A suboptimal cleavage site that is recognized by the NIa proteinase is located between the two domains. This site was found to be utilized in the VPg-associated, but not the nuclear, pool of NIa. A mutation converting Glu-189 to Leu at the P1 position of the processing site inhibited internal cleavage. Introduction of this mutation into TEV-GUS, an engineered variant of TEV that expresses a reporter protein (beta-glucuronidase [GUS]) fused to the N terminus of the helper component-proteinase (HC-Pro), rendered the virus replication defective in tobacco protoplasts. Site-specific reversion of the mutant internal processing site to the wild-type sequence restored virus viability. In addition, the trans-processing activity of NIa proteinase was tested in vivo after introduction of an artificial cleavage site between the GUS and HC-Pro sequences in the cytoplasmic GUS/HC-Pro polyprotein encoded by TEV-GUS. The novel site was recognized and processed in plants infected by the engineered virus, indicating the presence of excess NIa processing capacity in the cytoplasm. The potential roles of internal NIa processing in TEV-infected cells are discussed.     

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.     

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.     

An Ry-mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y

Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry-mediated resistance against PVY in potato, we expressed each of the PVY-encoded proteins in leaves of PVY-resistant (Ry) and -susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry-mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry-mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host-encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry-mediated resistance response.     

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Structure of arterivirus nsp4. The smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole

Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity.     

Molecular modeling and chemical modification for finding peptide inhibitor against severe acute respiratory syndrome coronavirus main proteinase

Severe acute respiratory syndrome (SARS) is a respiratory disease caused by a newly found virus, called SARS coronavirus. In this study, the cleavage mechanism of the SARS coronavirus main proteinase (Mpro or 3CLpro) on the octapeptide NH2-AVLQ downward arrowSGFR-COOH was investigated using molecular mechanics and quantum mechanics simulations based on the experimental structure of the proteinase. It has been observed that the catalytic dyad (His-41/Cys-145) site between domains I and II attracts the pi electron density from the peptide bond Gln-Ser, increasing the positive charge on C(CO) of Gln and the negative charge on N(NH) of Ser, so as to weaken the Gln-Ser peptide bond. The catalytic functional group is the imidazole group of His-41 and the S in Cys-145. Ndelta1 on the imidazole ring plays the acid-base catalytic role. Based on the "distorted key theory" [K.C. Chou, Anal. Biochem. 233 (1996) 1-14], the possibility to convert the octapeptide to a competent inhibitor has been studied. It has been found that the chemical bond between Gln and Ser will become much stronger and no longer cleavable by the SARS enzyme after either changing the carbonyl group CO of Gln to CH2 or CF2 or changing the NH of Ser to CH2 or CF2. The octapeptide thus modified might become an effective inhibitor or a potential drug candidate against SARS.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis

A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics. The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other. The entire glutathione molecule fits well into the cleft. A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66. Hydrogen bonds can be formed between Met-65 and the glutathione cysteine. This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide. In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16. Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80. The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein. Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity. Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency. A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant. Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.     

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.     

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease--crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 A resolution

Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.