The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.     

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.     

Binding of terbium and of an elastase inhibitor to bovine pancreatic subunit III, an inactive protease E

Using the Tb3+ luminescence technique, we showed that bovine subunit III, a defective pancreatic serine endopeptidase-like protease, possessed a single metal ion binding site able to bind Tb3+ with a high affinity comparable to that of porcine elastase. The topology of the metal ion binding site in subunit III is predicted from sequence homologies and modeling experiments based on the known crystallographic three-dimensional structures of the equivalent sites in porcine elastase 1 and bovine beta-trypsin. Moreover, the Tb3+ luminescence technique in parallel to a 19F NMR investigation, allowed us to measure the binding of a very potent specific inhibitor of porcine elastase (trifluoroacetyl-L-lysyl-alanyl p-trifluoromethylphenylanilide) to bovine subunit III. These results confirm that, although devoid of any specific activity, subunit III might possess a conformation close to that of an active enzyme and further support the analogy between subunit III and an elastase-like family.     

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display

The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group-'substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, k(cat)/K(M), of 2800 s(-1) M(-1), that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.     

A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations

Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process.     

The amino-terminal sequence of the catalytic subunit of bovine enterokinase

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.     

The crystal structure of guamerin in complex with chymotrypsin and the development of an elastase-specific inhibitor

Guamerin, a canonical serine protease inhibitor from Hirudo nipponia, was identified as an elastase-specific inhibitor and has potential application in various diseases caused by elevated elastase concentration. However, the application of guamerin is limited because it also shows inhibitory activity against other proteases. To improve the selectivity of guamerin as an elastase inhibitor, it is essential to understand the binding mode of the inhibitor to elastase and to other proteases. For this purpose, we determined the crystal structure of guamerin in complex with chymotrypsin at 2.5 Å resolution. The binding mode of guamerin on elastase was explored from the model structure of guamerin/elastase. Guamerin binds to the hydrophobic pocket of the protease in a substrate-like manner using its binding loop. In order to improve the binding selectivity of guamerin to elastase, several residues in the binding loop were mutated and the inhibitory activities of the mutants against elastase and chymotrypsin were monitored. The substitution of the Met36 residue for Ala in the P1 site increased the inhibitory activity against elastase up to 14-fold, while the same mutant showed 7-fold decreased activity against chymotrypsin compared to the wild-type guamerin. Furthermore, the M36A guamerin mutant more effectively protected endothelial cells against cell damage caused by elastase than the wild-type guamerin.     

Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences.

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and alpha-lytic protease have been compared with those of the pancreatic enzymes alpha-chymotrypsin and elastase. This comparison shows that approximately 60% (55-64%) of the alpha-carbon atom positions of the bacterial serine proteases are topologically equivalent to the alpha-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is approximately 84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacteria and pancreatic enzymes without significant primary sequence identity (less than 21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164-182) adopts very different comformations which are associated with their altered substrate specificities.     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships.

The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.     

Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B,VesB

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na⁺ did not stimulate the activity of VesB, despite containing the Tyr²⁵⁰ signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.     

Subunit III of bovine procarboxypeptidase A: its relationship to human pancreatic elastase 1

This paper is a continuation of our study of various animal pancreatic enzymes which are related to human pancreatic elastase 1 (Sziegoleit, A. & Linder, D. (1986) Biol. Chem. Hoppe-Seyler, 367, 527-531). The isolation and immunological analysis of the related protein from bovine pancreas disclosed that the third subunit of the procarboxypeptidase A complex is the antibody-binding component. The similarity of this subunit to elastase 1 is affirmed by comparison of their primary structures. While the complete amino-acid sequence of bovine subunit III recently has been published (Venot, N., Sciaky, M., Puigserver, A., Desnuelle, P. & Laurent, G. (1986) Eur. J. Biochem. 157, 91-99), we here present the amino-acid sequence of the carboxy-terminal tryptic peptide of human pancreatic elastase 1 showing a high degree of homology.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Modeling zymogen protein C

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.     

Complete amino acid sequence of streptokinase and its homology with serine proteases

The complete amino acid sequence of streptokinase has been determined by automated Edman degradation of its cyanogen bromide and proteolytic fragments. The protein consists of 415 amino acid residues. Sequence microheterogeneity was found at two positions. The NH2-terminal 245 residues of streptokinase are homologous to the sequences of several serine proteases including bovine trypsin and Streptomyces griseus proteases A and B. The sequence alignment suggests that the active-site histidine-57 has changed to a glycine in streptokinase. The other active-site residues, aspartyl-102 and serine-195, are, however, present at the expected positions. Streptokinase also contains internal sequence homology between the NH2-terminal 173 residues and a COOH-terminal 162-residue region between residues 254 and 415. Moderate homology in predicted secondary structures also exists between these two regions. Although streptokinase is not a protease, these observations suggest that it has evolved from a serine protease by gene duplication and fusion. A COOH-terminal region of about 80 residues is apparently deleted from the second half of the duplicated structures. These observations further suggest that the three-dimensional structure of streptokinase likely contains two independently folded domains, each homologous to serine proteases.     

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 Å resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.     

The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.