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The effect of a structural water molecule on the electronic nature of the His64-Asp32 hydrogen bond in subtilisins is examined by DFT calculations; the structural water is found to favor a short strong hydrogen bond in the catalytic triad in sharp contrast to some current beliefs.
We describe the development and validation of a portable system comprising an air sampler coupled to an automated flow injection analysis device. The system is able to monitor airborne concentrations of subtilisin-type enzymes in the workplace atmosphere on a continuous basis. Sampling is in two stages: using a sampling head that is designed to mimic human respiration at approx. 1 m s(-1) at a sampling rate of 600 l min(-1). In the second stage, the captured particles are deposited by impaction from the air stream onto the inner surface of a cyclone that is continuously washed with a jet of buffer solution. Deposited particles are then washed into a reservoir from which samples are taken every 5-6 min and injected automatically into a continuous flow injection analysis system. Proteolytic enzyme in the sample passes through a bioreactor maintained at about 40 degrees C. This contains a cellulose solid phase matrix on which is covalently immobilised Texas Red-labelled gelatin as substrate. The passing enzyme partially digests the substrate releasing fluorophore that is detected down stream in a flow cell coupled to a fluorimeter. The system is calibrated using enzyme standards and the intensity of the resulting peaks from the ex-air samples is converted to airborne concentrations using a mathematical model programmed into a PC. The system has a limit of detection of 4.8 ng m(-3) and a dynamic range of 5-60 ng m(-3). The within assay precision (RSD) is 6.3-9.6% over this range. The within batch precision is 20.3% at 20 ng m(-3) and the corresponding between batch value is 19.5%. The system has been run for periods up to 8 h in the laboratory and for up to 4 h at a factory site and the values obtained compared with time-averaged values obtained from a conventional Galley sampler and in-house analysis when reasonable agreement of the results was observed. The stability of the system over 21 days of continuous use with standards injected periodically was studied. Linearity was observed for all the standard plots throughout. At the end of 21 days, after a total exposure equivalent to 2395 ng ml(-1) of Savinase, the signal due to the 5.0 ng ml(-1) standard was still easily detectable.
Limited subtilisin digestion of the high-Mr haemoglobin of the crustacean Artemia sp. results in a series of fragments that are multiples of Mr 16000. Properties such as amino acid composition, iron content, absorption and c.d. spectra of the 16000-Mr functional units strongly resemble those of the intact haemoglobin molecules. The 16000-Mr functional units can bind O2 in a non-co-operative way. They thus represent the structural units from which the globin chains are built up.
PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.
The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.
The ale gene specifying the subtilisin YaB produced by alkalophilic Bacillus YaB, has an unusual start codon UUG. Changing this codon to AUG and GUG increased expression of the ale gene in B. subtilis DB104 and in an ale deficient mutant strain YaB-DEC4. The relative translational efficiency order of the three initiation codons is AUG > GUG > UUG in B. subtilis DB104 and in YaB-DEC4. These data suggest that the preferred initiation codon is AUG for ale gene expression in Bacillus.
An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.
The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.
Conformational energy calculations are used to analyze the interactions of structural substructures in subtilisin BPN. These substructures are kept fixed or "rigid" so that the only variables in the calculations are the backbone segments that separate them. The flexible segments are assumed to be free turns. Using this representation of the protein it is possible to predict both a likely order of events along a folding pathway and preferred modes of conformational changes of the native protein. Moreover, when the native structure has been perturbed by moving the substructures apart, it is possible to assess the range of interactions that return the protein, upon energy minimization, to its original conformation. These results suggest an approach to the folding problem based on the piecemeal formation of tertiary structure from smaller prefolded fragments.
The gelatin-binding region of fibronectin has been obtained by subtilisin digestion and cyanogen bromide cleavage of the molecule. Enzymatic digestion yielded two fragments of molecular weights 50,000 (S50K) and 30,000 (S30K) which were isolated by elution from gelatin-Sepharose affinity columns. Because the S50K fragment also mediated the adhesion of fibroblasts to collagen, it contains both the collagen and cell binding sites on the fibronectin molecule. Both fragments had valine as the NH2-terminal residue, were enriched in half-cystine and methionine residues compared to the whole molecule, and were identical by immunodiffusion. The S50K fragment begins with the sequence Val-Tyr-Gln-Pro-Gln-Pro-His-Pro-Gln-Pro-(Pro)-(Gly)-Tyr-Gly-His-( )-Val, a region with an extended conformation which is susceptible to proteolysis and connects this domain to the remainder of the fibronectin molecule. The S50K fragment appears to be located in the COOH-terminal one-third of the fibronectin molecule but does not contain the interchain disulfide bridge(s); the S30K fragment is probably derived from the NH2-terminal region of S50K.
Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.
Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.
The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of approximately 150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded beta-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.
PC1 and PC2 are two important subtilisin-like prohormone convertases (PC) that undergo differential endoproteolytic processing steps and sequentially mediate proopiomelanocortin (POMC) processing. To investigate the structural elements directing the processing of different PCs, we constructed a series of mutant and chimeric PC proteins and expressed them in cell lines with different patterns of expression of endogenous PCs: AtT-20, hEK293, and hLoVo cells. The COOH-terminally truncated PC1 underwent efficient proregion cleavage and rapid secretion in all three cell lines, while proregion cleavage and secretion were completely blocked in an active-site mutant of PC1. The truncated PC1 produced dramatic changes in POMC processing in AtT-20 cells. PC2 with the potential oxyanion hole Asp residue changed to Asn was processed and altered several aspects of POMC processing in a manner similar to that of wild-type PC2. PC1 protein with its proregion substituted with that of furin was cleaved after its proregion, producing active PC1 enzyme. A similar furin/PC2 fusion protein underwent proregion cleavage at low efficiency. By contrast, when the proregions of PC1 and PC2 were substituted with one another, both fusion proteins failed to cleave the foreign prosequences, were unable to undergo oligosaccharide maturation, and remained in the ER. Although inactive PC mutants could theoretically function as dominant negatives, none interfered with the processing of endogenous active PCs or with POMC processing. We conclude that the COOH-terminal of PC1 plays an important role in the routing or storage of PC1, the proregions of these PC proteins are replaceable in a molecule-specific manner, removal of proregion is essential for routing and for endoproteolytic activity, and the role of the potential oxyanion hole in PC2 is still unclear.
Insulin-like growth factor I (IGF-I) is required for normal embryonic development and postnatal growth. Like most hormones and growth factors, IGF-I is synthesized as a proprotein that is converted to the mature form by endoproteolysis. Processing of pro-IGF-I to mature IGF-I occurs by cleavage within the unique pentabasic processing motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg77. We have previously shown that human embryonic kidney 293 cells process pro-IGF-IA at Arg71 to generate IGF-I-(1-70) and at Arg77 to produce IGF-I-(1-76). Cleavage at each of these sites requires upstream basic residues, indicating that subtilisin-related proprotein convertases (SPCs) may be involved. In order to investigate the identity of the endogenous enzymes involved in maturation of pro-IGF-IA, we have expressed wild-type and mutant pro-IGF-IA in 293 cells and in the furin-deficient Chinese hamster ovary cell line, RPE.40. We have also co-expressed these constructs with SPCs that are thought to play a role in processing precursor proteins in the constitutive pathway: furin, PACE4, PC6A, PC6B, and LPC. The results show that furin is most active at cleaving wild-type and mutant pro-IGF-IA and can cleave these precursors at multiple sites within the pentabasic motif. PC6A and LPC are less active than furin but cleave only at Arg71. PACE4 and PC6B have very little activity on pro-IGF-IA precursors. Wild-type pro-IGF-IA was correctly processed to mature IGF-I in 10 of 10 cell lines that were tested. Since furin, PC6A, and LPC are known to have a broad pattern of tissue distribution and we have demonstrated expression of LPC in RPE.40 cells, our results suggest that these SPCs may be responsible for the endogenous pro-IGF-IA processing activity observed in a wide variety of cell lines.
A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and alpha-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in alpha-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.
In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). The pfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.
Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.