Oxyanion Hole Research Articles

Synthesis, biochemical evaluation, and molecular modeling studies of aryl and arylalkyl di-n-butyl phosphates, effective butyrylcholinesterase inhibitors

A series of dialkyl aryl phosphates and dialkyl arylalkyl phosphates were synthesized. Their inhibitory activities were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The di-n-butyl phosphate series consistently displayed selective inhibition of BChE over AChE. The most potent inhibitors of butyrylcholinesterase were di-n-butyl-3,5-dimethylphenyl phosphate (4b) [KI=1.0±0.4μM] and di-n-butyl 2-naphthyl phosphate (5b) [KI=1.9±0.4μM]. Molecular modeling was used to uncover three subsites within the active site gorge that accommodate the three substituents attached to the phosphate group. Phosphates 4b and 5b were found to bind to these three subsites in analogous fashion with the aromatic groups in both analogs being accommodated by the “lower region,” while the lone pairs on the PO oxygen atoms were oriented towards the oxyanion hole. In contrast, di-n-butyl-3,4-dimethylphenyl phosphate (4a) [KI=9±1μM], an isomer of 4b, was found to orient its aromatic group in the “upper left region” subsite as placement of this group in the “lower region” resulted in significant steric hindrance by a ridge-like region in this subsite. Future studies will be designed to exploit these features in an effort to develop inhibitors of higher inhibitory strength against butyrylcholinesterase.

An investigation into the applicability of the semiempirical method PM7 for modeling the catalytic mechanism in the enzyme chymotrypsin

The catalytic cycle for the serine protease α-chymotrypsin was investigated in an attempt to determine the suitability of using the semiempirical method PM7 in the program MOPAC for investigating enzyme-catalyzed reactions. All six classical intermediates were modeled using standard methods, and were characterized as stable minima on the potential energy surface. Using a modified saddle point optimization method, five transition states were located and verified both by vibrational and by intrinsic reaction coordinate analysis. Some individual features, such as the hydrogen bonds in the oxyanion hole, the nature of various electrostatic interactions, and the role of Met192, were examined. This involved designing and running computational experiments to model mutations that would allow features of interest, in particular the energies involved, to be isolated. Three features within the enzyme were examined in detail: the reaction site itself, where covalent bonds were made and broken, the electrostatic effects of the buried aspartate anion, a passive but essential component of the catalytic triad, and the oxyanion hole, where hydrogen bonds help stabilize charged intermediates. With one minor exception, all phenomena investigated agreed with previously-reported descriptions. This result, along with the fact that all the techniques used were relatively straightforward, leads to the recommendation that PM7 and related methods, such as PM6-D3H4, are appropriate for modeling similar enzyme-catalyzed reactions.Graphical abstractFifth of six transition states, showing water splitting into hydroxyl anion and a proton, to form the second tetrahedral intermediate and histidinium ion. Atoms of the water molecule involved in the hydrolysis are indicated by halos.

Structural Insights into Oxalate Biosynthetic Component

Bacterial quorum sensing (QS), a cell‐to‐cell communication process in many Proteobacteria, controls the gene expression for bacterial population‐wide characteristics, including bioluminescence, motility, and virulence‐related factors. Recently, QS has been recognized to provide further benefits at the population level by regulating the production of public goods, the function of which could be beneficial to all members of the group. In Burkholderia species, oxalic acid was recently identified as an excreted public good for the QS‐dependent growth. In these species, QS‐mediated oxalogenesis via the oxalate biosynthetic component (obc) is a cellular event indispensable for the survival of bacteria in the stationary phase. In B. glumae, obc consists of two genes encoding ObcA and ObcB for coordinating the production of oxalic acid, as well as acetoacetate and CoA, by using oxaloacetate and acetyl‐CoA as substrates. Unlike B. glumae, there is a bi‐functional enzyme Obc1 from B. thailandensis. Sequence analysis indicates that Obc1 has an ObcA‐like N‐terminal domain and shows ObcB activity in its C‐terminal domain despite no sequence homology with ObcB. We will present our recent progress on structural and functional analyses of ObcA and Obc1. In ObcA, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr322 as a general base, which then attacks the thioester carbonyl carbon of acetyl‐CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal‐coordination shell and absence of general acid(s) likely produces an unusual tetrahedral CoA adduct as a stable product. In Obc1, the C‐terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His‐934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis.Support or Funding InformationThis work was supported by Next Generation BioGreen 21 program of Rural Development Administration (Plant Molecular Breeding Center) of Republic of KOREA.

Structural insights into human microsomal epoxide hydrolase by combined homology modeling, molecular dynamics simulations, and molecular docking calculations

A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble-based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720-730. © 2016 Wiley Periodicals, Inc.

A Computational Investigation into the Mechanism of the Histone Acetyltransferase, Gcn5

Post-translational modifications (PTMs) can have a profound effect on protein structure and function. One such PTM is the acetylation of histone (a protein involved in DNA binding). In this reaction, an enzyme catalyzes the transfer of the acetyl group from acetyl CoA to a free lysine on the histone. This transfer neutralizes the positively charged lysine, which ultimately allows the DNA to be exposed for transcription. In our study, we focus on the acetyltransferase, Gcn5. Details regarding the reaction mechanism used by Gcn5 remain obscured. However, current mechanistic hypotheses suggest that the reaction occurs through a tetrahedral oxyanion intermediate, which is stabilized by a hydrogen bond to a nearby residue, i.e. an oxyanion hole. We utilize molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations in order to further probe possible mechanistic schemes of this reaction.

Mechanistic Insights into Ubc13-Catalyzed Ubiquitination

Ubc13 is an E2 enzyme that catalyzes lysine ubiquitination, a type of protein post-translation modification. Ubiquitinating a protein can signal for its degradation and affect its activity. Ubiquitination also plays a role in DNA repair and inflammatory response. Defects in this process are linked to different disorders including cancer, Parkinson's and Alzheimer's diseases. The accepted mechanism for Ubc13-catalyzed ubiquitination is a stepwise pathway that proceeds through an oxyanion intermediate. This intermediate is hypothesized to be stabilized by a nearby asparagine residue, which is known as the “oxyanion hole.” However, recent experimental results on mutated Ubc13 have suggested an alternate role for the asparagine. In our study, we use a combination of simulation techniques on the wild-type and mutated Ubc13 to examine its catalytic mechanism. Our calculations indicate that several different intermediates are possible, that water may stabilize the intermediate, and that the asparagine serves to stabilize a random coil near the active site.

Crystal Structure of StnA for the Biosynthesis of Antitumor Drug Streptonigrin Reveals a Unique Substrate Binding Mode

Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10′-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1.

Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.

De novo endo-functionalized organic cages as cooperative multi-hydrogen-bond-donating catalysts.

Two endo-functionalized organic cages as oxyanion hole mimics were achieved via dynamic covalent chemistry, which exhibit good size selectivity, catalytic activity and broad substrate scopes for Friedel-Crafts reactions. The modular character of the synthesis provides an easy way to modify the size, shape and inner function of the molecular cavity.

A cleft type receptor which combines an oxyanion hole with electrostatic interactions.

A receptor for carboxylic acids which combines an oxyanion-hole structure with electrostatic forces has been prepared. X-ray diffraction studies have been carried out to evaluate the geometry of both the free receptor and its associated species with several carboxylic acids and many different arrangements have been discovered for the H-bond pattern in the associated species.

A molecular receptor selective for zwitterionic alanine.

A molecular receptor has been synthesized joining an aza-crown ether with a chiral chromane which mimics the oxyanion hole of the enzymes. With this receptor an apolar host-guest complex with zwitterionic alanine has been achieved through the formation of up to seven H-bonds. This complex allows the extraction of aqueous alanine to a chloroform phase, while other natural amino acids are poorly extracted or are not extracted at all. Due to the chiral nature of the receptor, enantioselective extraction from the aqueous alanine solution to a chloroform phase takes place. X-Ray analysis combined with anisotropic effects, NOE and CD studies revealed the absolute configuration of both strong and weak complexes. Modelling studies also support the proposed structures. The presence of an oxyanion-hole motif in this structure was corroborated by X-ray diffraction studies.

A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII).

Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL β-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.

A Pitcher-and-Catcher Mechanism Drives Endogenous Substrate Isomerization by a Dehydrogenase in Kynurenine Metabolism

Aldehyde dehydrogenase typically performs oxidation of aldehydes to their corresponding carboxylic acid while reducing NAD(P)+ to NAD(P)H via covalent catalysis mediated by an active-site cysteine residue. One member of this superfamily, the enzyme 2-aminomuconate-6-semialdehyde dehydrogenase (AMSDH), is a component of the kynurenine pathway, which catabolizes tryptophan in mammals and certain bacteria. AMSDH catalyzes the NAD+-dependent oxidation of 2-aminomuconate semialdehyde to 2-aminomuconate. We recently determined the first crystal structure of AMSDH and several catalytic cycle intermediates. A conserved asparagine in the oxyanion hole, Asn-169, is found to be H-bonded to substrate-derived intermediates in the active site of AMSDH during catalysis, including both the covalently bound thiohemiacetal and thioacyl intermediates. To better interrogate the significance of the hydrogen bond provided by Asn-169 to the reaction mechanism of AMSDH, we created Ala, Ser, Asp, and Gln mutants and studied them using biochemical, kinetic, crystallographic, and computational studies. The in crystallo chemical reaction of the primary substrate with the co-crystalized complex of the N169D mutant and NAD+ led to the successful trapping of a new catalytic intermediate that was not previously seen. The structural and computational data are consistent with a substrate imine/enol tautomer intermediate being formed prior to the formation of the covalent bond between the substrate and the active-site cysteine. Thus, AMSDH surprisingly includes an isomerization process within its known catalytic mechanism. These data establish a hidden intrinsic isomerization activity of the dehydrogenase and allow us to propose a pitcher-catcher type of catalytic mechanism for the isomerization.

Insights from molecular dynamics on substrate binding and effects of active site mutations in Δ1-pyrroline-5-carboxylate dehydrogenase

The NAD+-dependent enzyme, Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both l-glutamate-γ-semialdehyde (GSA) and 4-erythro-hydroxy-l-glutamate-γ-semialdehyde (4-OH-GSA) to their respective l-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD+, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. Our in silico mutation analysis indicated that the conserved Glu447 has significantly shifted in both the S352L and E314A mutants, causing NAD+ to be displaced from its predictive orientation in the binding site and hence forming a catalytically inactive enzyme. However in the case of S352A, the catalytic site including the oxyanion hole and Cys348 remain virtually unchanged, and the coenzyme maintains its binding position.

Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source

Hydrazidase was an enzyme that remained unidentified for a half century. However, recently, it was purified, and its encoding gene was cloned. Microbacterium sp. strain HM58-2 grows with acylhydrazides as its sole carbon source; it produces hydrazidase and degrades acylhydrazides to acetate and hydrazides. The bacterial hydrazidase belongs to the amidase signature enzyme family and contains a Ser-cisSer-Lys catalytic motif. The condensation of hydrazine and carbonic acid produces various hydrazides, some of which are raw materials for synthesizing pharmaceuticals and other useful chemicals. Although natural hydrazide compounds have been identified, the metabolic systems for hydrazides are not fully understood. Here, we report the crystal structure of hydrazidase from Microbacterium sp. strain HM58-2. The active site was revealed to consist of a Ser-cisSer-Lys catalytic triad, in which Ser179 forms a covalent bond with a carbonyl carbon of the substrate. 4-Hydroxybenzoic acid hydrazide bound to the S179A mutant, showing an oxyanion hole composed of the three backbone amide groups. Furthermore, H336 in the non-conserved region in the amidase family may define the substrate specificity, which was confirmed by mutation analysis. A wild-type apoenzyme structure revealed an unidentified molecule covalently bound to S179, representing a tetrahedral intermediate.

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2).

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

Solid-State 17O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases.

We report preparation, trapping, and solid-state 17O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]benzoyl-chymotrypsin, trans-o-methoxy-[17O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[17O]cinnamoyl-chymotrypsin. We show that both the 17O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental 17O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═17O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the 17O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of 17O as a useful nuclear probe in NMR studies of enzymes.

Cloning of two carboxylesterase cDNAs from the swimming crab Portunus trituberculatus: Molecular evidences for their putative roles in methyl farnesotae degradation.

The sesquiterpenoid methyl farnesoate (MF) is the unepoxidized form of insect juvenile hormone (JH) III, and is considered an equivalent of JH in crustaceans. Degradation of MF is similar to that of JH which occurs through ester hydrolysis by specific carboxylesterases (CXEs). In this study, the full-length cDNAs of two JH esterase-like CXEs were cloned from the swimming crab, Portunus trituberculatus. The predicted amino acid sequences of the two PtCXEs contain the conserved motifs including catalytic triad and oxyanion hole, which are the hallmark of the CXE family proteins. The phylogenetic analysis showed that the two PtCXEs may belong to the hormone/semiochemical processing group of CXE family, indicating their possible roles on metabolism of hormones. Transcripts of both PtCXEs were most abundant in hepatopancreas and the PtCXE2 was also highly expressed in ovary. The mRNA levels of two PtCXEs in hepatopancreas were induced by in vivo MF treatment and eyestalk ablation, further indicating their potential in degrading MF. However, during the ovarian maturation, expression of the two PtCXEs increased significantly in the early-vitellogenic stage, prior to the remarkable rise in hemolymph MF titer reported by our previous studies. Taken together, our results suggest that the two PtCXEs can potentially serve as the MF esterases, but their catalytic activity may not be restricted to MF.

Crystal Structure of the Pseudomonas aeruginosa BEL-1 Extended-Spectrum β-Lactamase and Its Complexes with Moxalactam and Imipenem.

BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.

Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10.

Although kallikrein-related peptidase 10 (KLK10) is expressed in a variety of human tissues and body fluids, knowledge of its physiological functions is fragmentary. Similarly, the pathophysiology of KLK10 in cancer is not well understood. In some cancer types, a role as tumor suppressor has been suggested, while in others elevated expression is associated with poor patient prognosis. Active human KLK10 exhibits a unique, three residue longer N-terminus with respect to other serine proteases and an extended 99-loop nearly as long as in tissue kallikrein KLK1. Crystal structures of recombinant ligand-free KLK10 and a Zn2+ bound form explain to some extent the mixed trypsin- and chymotrypsin-like substrate specificity. Zn2+-inhibition of KLK10 appears to be based on a unique mechanism, which involves direct binding and blocking of the catalytic triad. Since the disordered N-terminus and several loops adopt a zymogen-like conformation, the active protease conformation is very likely induced by interaction with the substrate, in particular at the S1 subsite and at the unusual Ser193 as part of the oxyanion hole. The KLK10 structures indicate that the N-terminus, the nearby 75-, 148-, and the 99-loops are connected in an allosteric network, which is present in other trypsin-like serine proteases with several variations.