Mechanism Of Serine Proteases Research Articles

Charge Relay Without Proton Transfer: Coupling of Two Short Hydrogen Bonds via Imidazole in Models of Catalytic Triad of Serine Protease Active Site.

A homologous series of 20 substituted alcohol-imidazole-acetate model complexes imitating the charge relay system in Ser-His-Asp catalytic triad of serine proteases is considered quantum-chemically. We show qualitatively that the geometries of alcohol-imidazole and imidazole-acetate short hydrogen bonds are strongly coupled via the central imidazole and such complexes are capable of effectively relaying the charge from acetate to alcohol moiety upon relatively small concerted proton displacements. We hypothesize an alternative catalytic mechanism of serine proteases that does not require two complete proton transfers or hydrogen bond breakage between Ser and His residues.

A new catalytic site functioning in antigen cleavage by H34 catalytic antibody light chain

The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.

PH and non-covalent ligand binding modulate Zika virus NS2B/NS3 protease binding site residues: Discoveries from MD and constant pH MD simulations

Zika virus (ZIKV) is a global health concern and has been linked to severe neurological pathologies. Although no medication is available yet, many efforts to develop antivirals and host cell binding inhibitors led to attractive drug-like scaffolds, mainly targeting the nonstructural NS2B/NS3 protease (NS2B/NS3pro). NS2B/NS3pro active site has several titratable residues susceptible to pH changes and ligand binding; hence, understanding these residues' protonation is essential to drug design efforts targeting the active site. Here we use in silico methods to probe non-covalent binding and its effect on pKa shifts of the active site residues on a ligand-free protease and with a non-peptidic competitive inhibitor (Ki =13.5 µM). By applying constant pH molecular dynamics, we found that the catalytic residues of the unbound NS2B/NS3pro achieved the protonation needed for the serine protease mechanism over the pH value of 8.5. Nevertheless, the protease in the holo state achieved this same scenario at lower pH values. Also, non-covalent binding affected the catalytic triad (H51, D75, and S135) by stabilizing their distances and interaction network. Thus, NS2B/NS3pro residues configuration for activity might be both pH-dependent and influenced by ligand binding. However, compound presence within the binding site destabilized the NS2B, interfering with the closed and active conformation necessary for substrate binding and catalysis. Our outcomes provide valuable insights into non-covalent inhibitor behavior and its effect on protease active site residues, impacting optimization and design of novel compounds. Communicated by Ramaswamy H. Sarma

Serine proteases: how did chemists tease out their catalytic mechanism?

Serine proteases are a family of enzymes that degrade a wide range of proteins and play vital roles in a variety of biological processes. Their mechanism of action involves several catalytic strategies that are common in enzymatic catalysis. In this article, we review the catalytic mechanism of serine proteases and how it was elucidated over the years. The artful application of chemical probes to dissect the mechanism of serine proteases provides excellent examples for teaching present-day students and scientists how to solve complex chemical and biological problems through chemical logic.

TNF-α and IL-17A induce the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes

BackgroundSerine proteases have important roles in skin barrier function and desquamation, and the aberrant expression or the dysfunction of serine proteases is associated with the pathogenesis of skin diseases. Serine protease activities are tightly regulated by serine proteases such as kallikrein-related peptidases (KLKs) and serine protease inhibitors such as lympho-epithelial Kazal-type related inhibitor (LEKTI). For a better understating of diseases’ pathogenesis, the regulation mechanism of serine proteases and the inhibitors’ expression in epidermal keratinocytes must be clarified. ObjectivesTo investigate the effects of the cytokines on the expression of LEKTI in epidermal keratinocytes. MethodsNormal human epidermal keratinocytes (NHEKs) were stimulated with panels of inflammatory cytokines. The expression of serine protease inhibitors was analyzed using quantitative real-time PCR and ELISA. LEKTI expression in normal human skin and lesions from psoriasis or atopic dermatitis (AD) were analyzed by immunohistochemically and tape-stripping. Trypsin- and chymotrypsin-like serine protease activities in culture supernatants were measured by using specific substrates. ResultsTNF-α and IL-17A significantly induced the expression of LEKTI in NHEKs. The immunohistochemical and tape-stripping analysis revealed that psoriatic skin lesions had higher LEKTI expression compared to normal skin and AD lesions. Trypsin- and chymotrypsin-like protease activities in the culture media were upregulated 3–5 days later but attenuated 6–7 days later period by these cytokines. ConclusionsIn epidermal keratinocytes, the Th1&Th17 cytokines TNF-α and IL-17A induce the expression of serine protease inhibitor LEKTI, and it might occur to suppress the increase in the serine protease activities under inflammation.

The discovery of proteases and intramembrane proteolysis

Proteases carry out a wide variety of physiological functions. This review presents a brief history of protease research, starting with the original discovery of pepsin in 1836. Following the path of time, we revisit how proteases were originally classified based on their catalytic mechanism and how chemical and crystallographic studies unravelled the mechanism of serine proteases. Ongoing research on proteases addresses their biological roles, small molecule inhibitors for therapeutic uses, and protein engineering to modify their activities. The discovery of intramembrane proteases is more recent, beginning with the discovery of site-2 protease in 1997. Since then, different mechanistic classes of intramembrane proteases have been characterized, and many of these act in regulated intramembrane proteolysis in signaling pathways. Furthermore, the rhomboid intramembrane proteases were discovered by genetic and biochemical experiments in Drosophila and then in human cells. Research on the intramembrane proteases is expanding, as their biological importance is recognized.

Simple Design of an Enzyme-Inspired Supported Catalyst Based on a Catalytic Triad

Summary Enzyme active sites afford an intricate interplay of functional groups to mediate complex organic and inorganic reactions. Many hydrolytic enzymes use a catalytic triad comprising three different functional residues—(Ser(-OH), Hist(-imidazole), Asp(-CO 2 H))—that catalyze the hydrolysis of numerous unique substrates. Inspired by this design, we have developed a simple one-step synthesis for preparing a new supported catalytic system in which the three reactive groups of the catalytic triad (alcohol, imidazole, and carboxylate) are incorporated into a single functional unit. These artificial active sites can be coupled to a solid-phase support (Merrifield resin) by copper(I)-catalyzed azide-alkyne cycloaddition "click chemistry," and their effectiveness as esterolysis catalysts was demonstrated. Furthermore, tuning the local hydrophobicity of the resin particles with an approach analogous to the native enzyme hydrophobic pocket increased the catalytic efficiency. Quantum mechanics and molecular dynamics computational modeling were used to probe the catalytic effect and suggested a concerted two-step mechanism and hydrophobic nanoenvironment similar to that of hydrolytic enzymes.

Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

BackgroundSerine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A). MethodsBy taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography. ResultsIn the structures obtained at low pH (pH4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified. ConclusionsThe analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide. General significanceThis work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.

Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller–Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state.

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

Yet more intramolecular cross-links in Gram-positive surface proteins

The surface of Gram-positive bacteria comprises a single membrane and, typically, a thick layer of cross-linked peptidoglycan that imparts strength and rigidity. Anchored to this cell wall are protein assemblies, such as pili, and surface proteins, such as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that act as surface adhesins. These proteins are critical not only for bacterial binding to host surfaces in the early stages of infection but also for biofilm formation and immune evasion. In a series of studies published since 2007, these cell surface proteins and protein assemblies have been shown to contain isopeptide and thioester bonds, highly unusual intramolecular covalent linkages between amino acid side chains. These cross-links have either a structure-stabilizing role or may be directly involved in adhesion. Using an elegant combination of structural biology and mass spectrometry, Kwon et al. (1) reveal in PNAS an ester bond as yet another covalent linkage in a putative MSCRAMM from the Gram-positive pathogen Clostridium perfringens . Remarkably, this ester bond, joining the side chains of a Thr and Gln residue, is equivalent to an unresolved acyl-enzyme intermediate, formed on an autocatalytic pathway that resembles the mechanism of serine proteases. Despite fundamental differences in the way they are encoded in bacterial genomes, following delivery/assembly at the cell surface, pili and many MSCRAMMs share important similarities. In such proteins, a series of repetitive domains (commonly referred to as stalk regions) are responsible for the length of the structure and allow an N-terminal region to be positioned away from the cell surface. At the C terminus, both pili and MSCRAMMs are anchored to the cell wall by sortases (2). Although many of the building blocks of Gram-positive surface proteins and pili bear some resemblance to their Gram-negative … [↵][1]1To whom correspondence should be addressed. E-mail: Mark.Banfield{at}jic.ac.uk. [1]: #xref-corresp-1-1

Elucidating the Substrate Preference of the Bi-Functional Repressor-Protease LexA

The rise of drug resistance in bacteria is a growing problem, and poses a formidable medical and scientific challenge. Mutations - the driving force behind evolution and drug-resistance - have long been thought to arise spontaneously in bacteria, leading to the pessimistic view that the rise of drug resistance is inevitable. However, the acquisition of mutations is in fact a regulated process, in part tied to the bacterial stress response or SOS pathway.The key regulatory protein governing the SOS pathway is the bi-functional repressor protease, LexA. In its basal state, LexA represses genes involved in the SOS response (SOS genes). When stimulated following genotoxic stress, LexA auto-proteolyzes via a serine protease mechanism at an internal cleavage region. Self-cleavage leads to de-repression of SOS genes, activating pro-mutagenic pathways, such as genetic recombination and error-prone DNA replication. This increased mutation rate, in turn, can promote the development of drug resistance under stress.Here we employ structural modeling, saturation mutagenesis, and kinetics to systematically elucidate substrate preference of the LexA enzyme in Pseudomonas aeruginosa. Our studies elucidate the key determinants for substrate recognition and demonstrate that overall, LexA is a relatively tolerant protease. Furthermore, this study reveals positions in the LexA protein that are amenable for labeling with fluorescent probes, which can be used for conformational studies and inhibitor screens. Understanding the enzymes that regulate mutation can lead to insights about the evolution of bacteria and potentially open up new avenues for combating the rise of drug resistance

Rezymogenation of active urokinase induced by an inhibitory antibody

An important regulatory mechanism of serine proteases is the proteolytic conversion of the inactive pro-enzyme, or zymogen, into the active enzyme. This activation process is generally considered an irreversible process. In the present study, we demonstrate that an active enzyme can be converted back into its zymogen form. We determined the crystal structure of uPA (urokinase-type plasminogen activator) in complex with an inhibitory antibody, revealing that the antibody 'rezymogenizes' already activated uPA. The present study demonstrates a new regulatory mechanism of protease activity, which is also an extreme case of protein allostery. Mechanistically, the antibody binds a single surface-exposed loop, named the autolysis loop, thereby preventing the stabilization of uPA in its active conformation. We argue that this autolysis loop is a key structural element for rezymogenation of other proteases, and will be a new target site for pharmacological intervention with serine protease activity.

The Catalytic Aspartate Is Protonated in the Michaelis Complex Formed between Trypsin and an in Vitro Evolved Substrate-like Inhibitor: A REFINED MECHANISM OF SERINE PROTEASE ACTION

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the Oδ2–Cγ bond appears to be a double bond, with Oδ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on Oδ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mFobs − DFcalc density above 2.5 σ next to Oδ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pKa of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pKa histidine theory.

Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.

New insight into catalytic mechanism of serine proteases from ultra-high resolution X-ray studies

New insight into catalytic mechanism of serine proteases from ultra-high resolution X-ray studies

Analysis of Catalytic Mechanism of Serine Proteases. Viability of the Ring-Flip Hypothesis

Quantum calculations are applied to the active site of serine proteases, including four specific residues and a water molecule, as well as a substrate and proton donors in the oxyanion hole. Residues are tethered to the protein backbone of an X-ray structure but otherwise allowed to move freely to their lowest energy positions. The viability of the ring-flip hypothesis, which proposes that a 180 degrees rotation of the His-57 imidazole ring facilitates the catalysis, is assessed by comparison of energies of configurations both before and after such a flip. Specifically considered is the contribution to catalysis of the Ser-214 residue and a water molecule that is observed in the active site. The calculations provide detailed information concerning the nature, geometry, and strength of hydrogen bonds that are formed within the active site at each stage of the enzymatic mechanism.

Short hydrogen bonds in the catalytic mechanism of serine proteases

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked alpha/beta-heterodimer, where the beta-chain of HGF (HGF beta) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the beta-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF beta crystal structure shows that V495 inserts into the "activation pocket" near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF beta domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF alpha-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.

A Method for Automatically Interpreting Mass Spectra of 18O-Labeled Isotopic Clusters

16O/18O labeling is one differential proteomics technology among many that promises diagnostic and prognostic biomarkers of disease. Although the incorporation of 18O in the C-terminal carboxyl group during endoproteinase digestion in the presence of H2 18O makes the process of labeling facile, the ease and effectiveness of label incorporation have in some regards been outweighed by the difficulties in interpreting the resulting spectra. Complex isotope patterns result from the composition of unlabeled (18O(0)), singly labeled (18O(1)), and doubly labeled species (18O(2)) as well as contributions from the naturally occurring isotopes (e.g. 13C and 15N). Moreover because labeling is enzymatic, the number of 18O atoms incorporated can vary from peptide to peptide. Finally it is difficult to distinguish highly up-regulated from highly down-regulated or C-terminal peptides. We have developed an algorithm entitled regression analysis applied to mass spectrometry (RAAMS) that automatically, rapidly, and confidently interprets spectra of 18O-labeled peptides without requiring chemical composition information derived from product ion spectra. The algorithm is able to measure the effective 18O incorporation rate due to variable enzyme substrate specificity of the pseudosubstrate during the isotope exchange reaction and corrects for the 18O(0) abundance that remains in the labeled sample when using a two-step digestion/labeling procedure. We have also incorporated a method for distinguishing pure 18O(0) from pure 18O(2) peptides utilizing impure H2 18O. The algorithm operates on centroided peak lists and is therefore very fast: nine chromatograms of, on average, 1,168 spectra and containing, on average, 6,761 isotopic clusters were interpreted in, on average, 45 s per chromatogram. RAAMS is fast enough (average, 38 ms/spectrum) to allow the possibility of performing information-dependent MS/MS on a chromatographic time scale on species exceeding predetermined ratio thresholds. We describe in detail the operation of the algorithm and demonstrate its use on datasets with known and unknown ratios.