Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the ß-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the ß-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

The comparative analysis of the properties and structures of purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27.

Two recombinant purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27 encoded by genes TT_C1070 (TthPNPI) and TT_C0194 (TthPNPII) were purified and characterized. The comparative analysis of their sequences, molecular weight, enzymes specificity and kinetics of the catalyzed reaction were realized. As a result, it was determined that the TthPNPI is specific to guanosine while the TthPNPII to adenosine. According to the results of the size exclusion chromatography and SAXS study both enzymes are hexameric molecules. Based on the sequence alignment with homologous purine nucleoside phosphorylases (PNPs), Asn was identified as a purine base recognizing residue in the active site of TthPNPI and Asp in TthPNPII. The three-dimensional structure of TthPNPII was solved at 2.5 Å resolution by molecular replacement method using crystals grown in microgravity. Position of phosphate in the active site cavity is located. The possible arrangement of adenosine and guanosine in TthPNPII active site cavity is considered using superposition with the structures of homologous trimeric and hexameric PNPs complexed with corresponding substrates. The peculiarities of oligomeric structure of TthPNPII in comparison with homologous PNPs are described. It is shown that two trimeric molecules of TthPNPII in the asymmetric part of the unit cell are connected by three two-fold axis into a hexamer with 32-point symmetry. This type of hexameric structure of PNP is found for the first time. The interface area between the subunits in trimeric molecule and between the trimers in TthPNPII hexamer is described.Communicated by Ramaswamy H. Sarma.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine.

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme.

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl2 and CuCl2) as precipitants and the three-dimensional structures were determined at 1.35 Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu+2, Ni+2 and Na+1 cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.Communicated by Ramaswamy H. Sarma.

The nature of the ligand's side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex.

The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp3-hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of CPB, the relative orientation of the catalytic amino acid residues, as well as the distance between Glu270 and SArg/SPhe, is much less dependent on the nature of the corresponding side chain of the substrate. The influence of the nature of the substrate side chain on the structural organization of the transition state determines catalytic activity and broad substrate specificity of the carboxypeptidase T.

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1' subsite from pancreatic carboxypeptidase B.

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Šresolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with acyclovir.

Escherichia coli purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family I hexameric PNPs. Owing to their key role in the purine salvage pathway, PNPs are attractive targets for drug design against some pathogens. Acyclovir (ACV) is an acyclic derivative of the PNP substrate guanosine and is used as an antiviral drug for the treatment of some human viral infections. The crystalline complex of E. coli PNP with acyclovir was prepared by co-crystallization in microgravity using counter-diffusion through a gel layer in a capillary. The structure of the E. coli PNP-ACV complex was solved at 2.32 Šresolution using the molecular-replacement method. The ACV molecule is observed in two conformations and sulfate ions were located in both the nucleoside-binding and phosphate-binding pockets of the enzyme. A comparison with the complexes of other hexameric and trimeric PNPs with ACV shows the similarity in acyclovir binding by these enzymes.

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51 Šresolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6122, with unit-cell parameters a = b = 120.370, c = 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Crystal structure of recombinant phosphoribosylpyrophosphate synthetase 2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions.

Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strain Thermus thermophilus HB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Šresolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits in T. thermophilus PRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic ß-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.

Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-arginine.

Porcine pancreatic carboxypeptidase B (EC 3.4.23.6) was complexed with a stable transition-state analogue, N-sulfamoyl-L-arginine, in which an S atom imitates the sp(3)-hybridized carbon in the scissile-bond surrogate. Crystals were grown in a form belonging to the same space group, P41212, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 Å. The molecule was refined and the positions of non-H atoms of the inhibitor and water molecules were defined using difference Fourier maps. The enzyme-inhibitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation between the complexed and uncomplexed forms of carboxypeptidase B are shown. The inhibitor is bound in a curved conformation in the active-site cleft, and the sulfamide group is bound to the Zn ion in an asymmetric bidentate fashion. The complex is stabilized by hydrogen bonds between the N1/N2 guanidine group of the inhibitor and the Asp255 carboxyl of the enzyme. The side-chain CH2 groups of the inhibitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may bind to carboxypeptidase B and therefore provides an insight into the structural basis of carboxypeptidase B selectivity, which is useful for the rational design of a carboxypeptidase with improved selectivity for industrial recombinant pro-insulin processing.

Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (ß-1,3-glucanase) Lic16A of Clostridium thermocellum.

The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacterium Clostridium thermocellum (ctCBM54) are reported. Recombinant ctCBM54 was prepared using an Escherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space group P6322, with unit-cell parameters a = b = 130.15, c = 131.05 Å. The three-dimensional structure of ctCBM54 will provide valuable information about the structure-function relation of the laminarinase Lic16A and will allow the exploitation of this binding module in biotechnological applications.

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 Å and 1.62 Å to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1' subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1' subsite and the catalytic center.

3'-Azidothymidine in the active site of Escherichia coli thymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study.

The structural study of complexes of thymidine phosphorylase (TP) with nucleoside analogues which inhibit its activity is of special interest because many of these compounds are used as chemotherapeutic agents. Determination of kinetic parameters showed that 3'-azido-3'-deoxythymidine (3'-azidothymidine; AZT), which is widely used for the treatment of human immunodeficiency virus, is a reversible noncompetitive inhibitor of Escherichia coli thymidine phosphorylase (TP). The three-dimensional structure of E. coli TP complexed with AZT was solved by the molecular-replacement method and was refined at 1.52 Šresolution. Crystals for X-ray study were grown in microgravity by the counter-diffusion technique from a solution of the protein in phosphate buffer with ammonium sulfate as a precipitant. The AZT molecule was located with full occupancy in the electron-density maps in the nucleoside-binding pocket of TP, whereas the phosphate-binding pocket of the enzyme was occupied by phosphate (or sulfate) ion. The structure of the active-site cavity and conformational changes of the enzyme upon AZT binding are described in detail. It is found that the position of AZT differs remarkably from the positions of the pyrimidine bases and nucleoside analogues in other known complexes of pyrimidine phosphorylases, but coincides well with the position of 2'-fluoro-3'-azido-2',3'-dideoxyuridine (N3FddU) in the recently investigated complex of E. coli TP with this ligand (Timofeev et al., 2013). The peculiarities of the arrangement of N3FddU and 3'-azidothymidine in the nucleoside binding pocket of TP and correlations between the arrangement and inhibitory properties of these compounds are discussed.

X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis during the catalyzed reaction.

Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis (PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Šresolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt-ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on C(α) atoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme-substrate and enzyme-product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

Crystal structure of the protealysin precursor: insights into propeptide function.

Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences ( approximately 200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short ( approximately 50 amino acids) propeptides. Here, we present the 1.8 A crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the "open" conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S' subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.

Processing of protealysin precursor.

Protealysin, a protease previously described by us in Serratia proteamaculans, belongs to the group of thermolysin-like proteases (TLPs) that differ from classical TLPs by the precursor structural organization. The propeptide of protealysin precursor has no significant structural similarity to the propeptides of most TLPs. The functions of protealysin-like precursors and mechanisms of their action remain unclear. We studied the pathway of protealysin precursor processing in vitro using standard approaches: modification of the catalytic site and monitoring immobilized precursor maturation. The Glu(113) --> Ala substitution inhibited the precursor maturation, which pointed to the autocatalytic processing. The mutant precursor exposure to active protealysin converted it to the mature enzyme, thus, indicating the intermolecular processing. Intermolecular processing of the mutant protein by other proteases such as thermolysin or subtilisin is also possible. The intact protealysin precursor was efficiently autoprocessed in solution but not after immobilization. These data indicate that the processing of protealysin precursor differs from that of classical TLPs. The protealysin propeptide is cleaved by an autocatalytic or heterocatalytic intermolecular mechanism and is most likely not removed intramolecularly.

The crystal structure of glutamyl endopeptidase from Bacillus intermedius reveals a structural link between zymogen activation and charge compensation.

Extracellular glutamyl endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsin-like serine protease which cleaves the peptide bond on the carboxyl side of glutamic acid. Its three-dimensional structure was determined for C222(1) and C2 crystal forms of BIEP to 1.5 and 1.75 A resolution, respectively. The topology of BIEP diverges from the most common chymotrypsin architecture, because one of the domains consists of a beta-sandwich consisting of two antiparallel beta-sheets and two helices. In the C2 crystals, a 2-methyl-2,4-pentanediol (MPD) molecule was found in the substrate binding site, mimicking a glutamic acid. This enabled the identification of the residues involved in the substrate recognition. The presence of the MPD molecule causes a change in the active site; the interaction between two catalytic residues (His47 and Ser171) is disrupted. The N-terminal end of the enzyme is involved in the formation of the substrate binding pocket. This indicates a direct relation between zymogen activation and substrate charge compensation.