Subtilisin Family Of Serine Proteases Research Articles

Structure-based discovery of small molecules improving stability of human broadly-neutralizing anti-HIV antibody 2F5 in plant suspension cells.

The production of biopharmaceuticals in engineered plant-based systems is a promising technology that has proven its suitability for the production of various recombinant glyco-proteins that are currently undergoing clinical trials. However, compared to mammalian cell lines, the productivity of plant-based systems still requires further improvement. A major obstacle is the proteolytic degradation of recombinant target proteins by endogenous plant proteases mainly from the subtilisin family of serine proteases. In the present study, the authors screened for putative small molecule inhibitors for subtilases that are secreted from tobacco BY-2 suspension cells using an in silico approach. The effectiveness of the substances identified in this screen was subsequently tested in degradation assays using the human broadly-neutralizing anti-HIV monoclonal antibody 2F5 (mAb2F5) and spent BY-2 culture medium as a model system. Among 16 putative inhibitors identified by in silico studies, three naphthalene sulfonic acid derivatives showed inhibitory activity in in vitro degradation assays and are similar to or even more effective than phenylmethylsulfonyl fluoride (PMSF), a classical inhibitor of serine proteases, which served as positive control.

Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism

A distinct class of the biologically important subtilisin family of serine proteases functions exclusively within the cell and forms a major component of the bacilli degradome. However, the mode and mechanism of posttranslational regulation of intracellular protease activity are unknown. Here we describe the role played by a short N-terminal extension prosequence novel amongst the subtilisins that regulates intracellular subtilisin protease (ISP) activity through two distinct modes: active site blocking and catalytic triad rearrangement. The full-length proenzyme (proISP) is inactive until specific proteolytic processing removes the first 18 amino acids that comprise the N-terminal extension, with processing appearing to be performed by ISP itself. A synthetic peptide corresponding to the N-terminal extension behaves as a mixed noncompetitive inhibitor of active ISP with a K(i) of 1μM. The structure of the processed form has been determined at 2.6Å resolution and compared with that of the full-length protein, in which the N-terminal extension binds back over the active site. Unique to ISP, a conserved proline introduces a backbone kink that shifts the scissile bond beyond reach of the catalytic serine and in addition the catalytic triad is disrupted. In the processed form, access to the active site is unblocked by removal of the N-terminal extension and the catalytic triad rearranges to a functional conformation. These studies provide a new molecular insight concerning the mechanisms by which subtilisins and protease activity as a whole, especially within the confines of a cell, can be regulated.

Structure Prediction of Neuroendocrine Convertase -2: A Potential Target in Various Cancers

Prohormone or proprotein convertases (PC2) are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues within the general motif (K/R)-(X)n- (K/R)2, where n is 0, 2, 4 or 6 and X is usually not Cys. Among the members of this prohormone convertase family, Neuroendocrine Convertase-2 (NEC-2) is regarded as one of the important proteins involved in the maturation of many precursor proteins. Being widely distributed in the neuroendocrine cells, these proteins play a vital role in causing malignant gliomas. They can serve as important drug targets in the treatment of cancers. In the present study, a 3D model of NEC-2 was generated using homology modeling. The model was optimized by a brief energy minimization in CHARMM and dynamics simulation of 250ps in MOE. The validation results of PROCHECK and Profile 3D show that the stereochemical quality of the model is good. The Calpha backbone of the template and the target (NEC-2) when superimposed showed RMSD of 0.39A. The model showed Asp51, His92 and Ser268 in the active site as seen in most of the PC2 members. The NEC-2 structure differs from that of furin at the catalytic pocket region with relevance to the amino acid composition which can be exploited for the design of specific inhibitors towards NEC-2.

Characteristic Features in the Structure and Collagen-Binding Ability of a Thermophilic Collagenolytic Protease from the Thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.

Microbial keratinases and their prospective applications: an overview

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide "keratin" recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.

Characterization of Endothelin-converting Enzyme-2: IMPLICATION FOR A ROLE IN THE NONCLASSICAL PROCESSING OF REGULATORY PEPTIDES

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.

Soybean subtilisin-like protease involved in initiating storage protein degradation

A partial cDNA fragment (1.1kb) of protease C1, an enzyme that initiates the proteolytic degradation of the β-conglycinin storage proteins in the soybean (Glycine max [L.] Merrill. cv. Amsoy 71) through limited action at Glurich regions, has been cloned by reverse transcription-polymerase chain reaction (RT-PCR). The sequence was extended toward the 5' end by another 314 bases. The nucleotide sequence shows that protease C1 belongs to the subtilisin family of serine proteases. The sequence encompasses the critical Asp, His and Ser residues of the catalytic triad, as well as the Asn at the binding site. Northern analysis shows the presence of a 2.5kb mRNA not only in seedling cotyledons, but also in developing seeds. The developing seeds, and even dry seeds, exhibit phenylmethylsulfonyl fluoride (PMSF) and EDTA-sensitive protease activity that only cleaves the α- and α'-, but not the β-subunits, of soybean β-conglycinin. The discrete proteolytic intermediates produced are of the same sizes as those produced by pure enzyme, as are the final 50 kDa and 48 kDa products. The activity is also sensitive to inhibition by synthetic poly-L-Glu, all characteristics of purified protease C1. These data suggest that protease C1, or an enzyme very similar to it, is synthesized in a form that is active in vitro. Because seeds do accumulate β-conglycinin and because there is very little evidence of the proteolytic products of protease C1 action in extracts of dry seeds, one can surmise that the protease C1 is not particularly active in vivo during seed maturation. GenBank lists the sequence of the 1.1kb fragment with accession number AAD02075.

Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei.

Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases.

Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei

Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50 241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases.

The occurrence of a tripeptidyl peptidase I in trophozoites of Entamoeba histolytica.

Tripeptidyl aminopeptidases are exopeptidases involved in intracellular protein degradation downstream of endopeptidases [for review, see (1)]. Mammalian tripeptide-releasing enzymes identified to date have been designated as tripeptidyl peptidases I (TPP I), which are localized in lysosomes, and tripeptidyl peptidases II (TPP II), which are cytoplasmatic. Additionally, evolutionarily unrelated TPPs have been identified in bacteria (1). Tripeptidases II and homologues belong to the subtilisin family of serine proteases and, like the proteasome, seem to have an ubiquitous distribution in eukaryotes, suggesting that they cooperate with the proteasome and with dipeptidases in degrading substrate proteins the entire way down into amino acids. Lysosomal TPP I, which further splits peptides generated by cathepsins, may also be a serine-type peptidase. Monomers of the enzyme, which generally are glycosylated, have a molecular mass between 48 kDa (human) and 107 kDa ( Dictyostelium ), and can form aggregates exhibiting molecular masses from 200–700 kDa (2). In addition to its role in lysosomal protein degradation, tripeptidase I has been suggested to be involved in more specific functions, such as generating signaling peptides or proteins for developmental processes (2). Entamoeba histolytica contains vesicular endopeptidases, the genes of which have been well characterized and have been shown to be cysteine proteinases (EhCP1, EhCP2, EhCP5, histolysain, and amebapain). Their activities contribute to lysosomal protein digestion and, extracellularly, to the attack of host structural proteins (3). The cytoplasm contains proteasomes that probably function in the endopeptidolytic cleavage of substrate proteins (4). By contrast, nothing is known about exopeptidases acting downstream of either cathepsins or of the proteasome in this parasite. Here, we report first data on a lysosomal/extracellular tripeptidyl peptidase I, with activity against AAF-X or AFP-X (X being a peptidolytically released fluorophor), in E. histolytica.

The exon-intron organization of the prohormone convertase PC2 gene from the insect Lucilia cuprina.

Prohormone or proprotein convertases are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues. Among the different members of this prohormone convertase family, the prohormone convertase 2 (PC2) is almost exclusively expressed in endocrine and neuroendocrine tissues and plays an important role in the endoproteolytic processing of prohormones. Here we describe the exon-intron organization of the PC2 gene from the insect Lucilia cuprina by characterization of PCR-amplified genomic DNA fragments. The insect PC2 gene contains 12 exons with an estimated size of over 14.5 kb. The exon sizes range from 38 bp to > 448 bp. All identified intron-exon boundaries are consistent with the GT-AG-rule. A comparison of the genomic structures of the thus far known prohormone convertase genes with that of the insect PC2 gene revealed a conservation of the positions of most introns interrupting the exons coding for the amino-terminal and catalytic domains. This conservation is consistent with the suggestion of a common evolutionary origin for the prohormone convertase gene family.

Isolation and characterization of insect PC2-like prohormone convertase cDNA.

Endoproteolytic processing of large precursor molecules at basic amino acid residues plays an important role in the maturation of many hormones, neuropeptides and other regulatory proteins. Enzymes performing these reactions are designated as prohormone or proprotein convertases and belong to the subtilisin family of serine proteases. The screening of a larval cDNA library of the sheep blowfly Lucilia cuprina resulted in the isolation of two cDNAs encoding a PC2-like prohormone convertase. The predicted 675 amino acid preproprotein (LcuPC2) exhibits its highest identity to invertebrate and vertebrate prohormone convertase 2 homologues, and a noticeably lower identity to the so far known insect furin-like prohormone convertases of Drosophila melanogaster and Aedes aegypti. In Northern blot experiments a signal at 2.5 kb could be detected.

Identification of Essential Residues for the Catalytic Function of 85-kDa Cytosolic Phospholipase A2: PROBING THE ROLE OF HISTIDINE, ASPARTIC ACID, CYSTEINE, AND ARGININE

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel.

Crystallographic study of eglin-C binding to thermitase.

Thermitase is a thermostable member of the subtilisin family of serine proteases isolated from Thermoactinomyces vulgaris l. It consists of a single polypeptide chain of 279 amino acid residues2. The degree of sequence homology to subtilisins BPN’ and Carlsberg is 42% and 44%, respectively. Eglin C is a serine protease inhibitor of 70 residues and is isolated from Hirudo medicinalis 3. The three-dimensional structure of eglin C has been determined previously in complex with subtilisin Carlsberg4.5. It was shown that the reactive loop of the inhibitor molecule noncovalently interacts with the enzyme at the binding groove. The inhibition effect of eglin C on subtilisins is ascribed to its ability to preserve the intact reactive loop via intensive intramolecular electrostatic interactions. Due to polypeptide nature, eglin C can be considered as a good model to study substrate recognition by subtilisins.

Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora

When grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular proteases hydrolysing the chromogenic substrate Azocoll. The protease activity was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PII immobilized P. redivivus in bioassays and hydrolysed proteins of the purified cuticle. The enzyme hydrolysed several protein substrates including casein, bovine serum albumin and gelatin, but not native collagen. Examination of substrate specificity with synthetic peptides showed that PII readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-Gly-Phe-NA). Mono-peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active site histidine residue of the subtilisin family of serine proteases.

Furin-mediated proprotein processing activity: Involvement of negatively charged amino acid residues in the substrate binding region

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neural residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

One-step site-directed mutagenesis of the Kex2 protease oxyanion hole

Background: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-α-factor, the precursor of the α-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the ‘oxyanion hole’.Results: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature α-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the kcal of the Asp 314 enzyme was reduced ∼ 4500-fold and its KM ∼4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-α-factor, however, kcat of the Asp 314 enzyme was reduced only 125-fold, while the Km was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo — by the intramolecular cleavage that removes its amino-terminal pro-domain — occurs at an unchanged rate.Conclusions: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer kcaf discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.

Presence of furin mRNA in cultured bovine endothelial cells and possible involvement of furin in the processing of the endothelin precursor.

Recently it was documented that furin, a calcium-dependent serine endoprotease, cleaves many protein precursors at pairs of basic amino acids, thus liberating the biologically active peptides. The endothelin precursors follow a biosynthetic pathway similar to these proteins, where the precursor is initially processed to the intermediate, big endothelin (big ET) before its conversion to the endothelin (ET) peptide. Analysis of the amino acid sequence of the endothelin pro-proteins shows that they are susceptible to processing by endoproteases that cleave at pairs of basic amino acids. For example, human endothelin-1 (ET-1) precursor possesses a typical furin cleavage site motif (Arg-X-Lys/Arg-Arg) at the following residues: Arg32-Ser33-Lys34-Arg35 and Arg72-Ser73-Lys74-Arg75. We have isolated mRNA from cultured bovine endothelial cells and, using a human furin cRNA probe, shown that a furin mRNA of 4.5 kb is present in these cells. We propose that furin, a novel endoprotease belonging to the mammalian subtilisin family of serine proteases, may be implicated in the processing of pro-endothelin precursors, liberating big ET.

Effects of eglin-c binding to thermitase: three-dimensional structure comparison of native thermitase and thermitase eglin-c complexes.

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin-c, grown from three different calcium concentrations. It appeared that the B-factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably similar to those of the 1.4 A native structure: the main chain atoms have an rms difference of only 2.3 A2; for all atoms this difference is 4.6 A2. The rms positional differences between these two structures of thermitase are 0.31 A for the main chain atoms and 0.58 A for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X-ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect. A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rms deviation of 0.13 A for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 A in C alpha positions. These are probably due to crystal packing effects. The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.

Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis

We have characterized glycoprotein H (GpH) from bovine adrenal medullary chromaffin granules. Two-dimensional gel electrophoresis was used to purify GpH from an insoluble fraction obtained following extraction of chromaffin granule membranes with lithium diiodosalicylate. The GpH material was recovered from two-dimensional gel spots by concentration and recovery on a one-dimensional gel followed by electro-blotting to a poly(vinylidene difluoride) membrane. This material was subjected to in situ tryptic digestion. The released peptides were purified by microbore high performance liquid chromatography and sequenced. The peptide sequences revealed extensive similarity to the mammalian kex2/subtilisin-related proteases (PC2 and PC3) which have been characterized recently by molecular cloning and sequence analysis (Smeekens, S. P., and Steiner, D. F. (1990) J. Biol. Chem. 265, 2997-3000; Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J., and Steiner, D. F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 340-344). The sequence similarity included regions that contain residues equivalent to the aspartic acid and histidine residues which are involved in the active site of the subtilisin family of serine proteases. The sequence data revealed the presence of tryptic peptides derived from both PC2 and PC3. NH2-terminal sequence analysis of GpH gave two sequences which were aligned with residues 110-121 of PC2 and PC3. It is likely that these sequences represent the mature form of PC2 and PC3 in chromaffin granules. These forms would be generated by cleavage at a site which is conserved in mammalian kex2-related enzymes and which would result in the release of approximately 80-residue propeptides. It was concluded that the spot identified as GpH by two-dimensional gel electrophoresis contains the bovine counterparts of both PC2 and PC3. The direct identification of these components in chromaffin granules supports their role in the processing of protein precursors.