Peptide Bond Hydrolysis Research Articles

N-Terminomics Strategies for Protease Substrates Profiling.

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

Nicking and fragmentation are responsible for α-lactalbumin amyloid fibril formation at acidic pH and elevated temperature.

Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble β-sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α-lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH1.6 and 65°C under non-reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non-reducing and reducing gel. We have identified the fragments by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins.

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.

Peptidyl Activity-Based Probes for Imaging Serine Proteases.

Proteases catalyze the hydrolysis of peptide bonds. Products of this breakdown mediate signaling in an enormous number of biological processes. Serine proteases constitute the most numerous group of proteases, accounting for 40%, and they are prevalent in many physiological functions, both normal and disease-related functions, making them one of the most important enzymes in humans. The activity of proteases is controlled at the expression level by posttranslational modifications and/or endogenous inhibitors. The study of serine proteases requires specific reagents not only for detecting their activity but also for their imaging. Such tools include inhibitors or substrate-related chemical molecules that allow the detection of proteolysis and visual observation of active enzymes, thus facilitating the characterization of the activity of proteases in the complex proteome. Peptidyl activity-based probes (ABPs) have been extensively studied recently, and this review describes the basic principles in the design of peptide-based imaging agents for serine proteases, provides examples of activity-based probe applications and critically discusses their strengths, weaknesses, challenges and limitations.

A novel “off-on-off” fluorescent sensor based on inner filter effect for ultrasensitive detection of protamine/trypsin and subcellular colocalization

Protease is an important physiological enzyme that can catalyze the hydrolysis of peptide bonds at specific sites and trypsin is a proteolytic enzyme present in the pancreas, the fluctuation of whom reflect dysfunction of body. Thus, it is necessary to develop sensitivity detections for protamine and trypsin. Herein, an "off-on-off" fluorescent sensor based on the inner filter effect was established for individually quantification of protamine and trypsin by using silicon quantum dots (SiQDs) and triangular silver nanoprisms (TSNPRs) based on the inner filter effect (IFE). The maximum absorption peak position of TSNPRs is 550 nm, which completely overlaps the emission peak (536 nm) of SiQDs and considerably improves sensitivity of the sensor. The proposed IFE-based sensor proved a good linearity from 0–30 ng/mL for protamine determination with detection limit of 5 ng/mL. For trypsin detection, the constructed sensor displayed a liner detection range from 0 to 40 ng/mL with a detection limit of 8 ng/mL. Besides, the SiQDs have equipped with the excellent capability for subcellular colocalization, providing a promising potential for practical applications.

Molecular identification of a new isolate of actinobacteria ATIS61 and characterization of the protease activities

Abstract. Amfar F, Fitri L, Suhartono. 2021. Molecular identification of a new isolate of actinobacteria ATIS61 and characterization of the protease activities. Biodiversitas 22: 1564-1569. Protease is an enzyme that catalyzes the hydrolysis of peptide bonds in protein. Actinobacteria are one of bacterial groups that is able to produce protease. Actinobacteria are Gram-positive bacteria and mostly aerobic. This study aimed to identify protease-producing actinobacteria ATIS61 isolate using 16S rRNA gene and to characterize the protease activity. This study was experimental research, consisted of amplification and sequencing of the 16S rRNA gene, and protease activity test. The 16S rRNA gene analysis showed that ATIS61 isolate was closely related to Nocardia sp. strain 335427 with a 99.88% similarity and the result of phylogenetic tree construction was related to Nocardia farcinica strain ARS8 with Bootstrap 94%. Protease activity test showed the highest activity was on the eighth day of incubation at 0.115 U/mL. Protease activity based on temperature showed the highest activity at 40°C of 0.156 U/mL and the stability of protease towards temperature was stable at 40°C and 50°C. Protease activity showed that the highest protease activity was at pH 8 of 0.096 U/mL and the highest protease stability was also at pH 8. The addition of HgCl2 showed that it could inhibit protease activity with a value of 0.059 U/mL.

Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis

A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

Insights into the Catalytic Mechanism of Domains CD1 and CD2 in Histone Deacetylase 6 from Quantum Calculations

The histone deacetylase 6 enzyme, HDAC6, is a metalloprotein with multiple pathophysiological roles and, as such, is the subject of intense research and drug discovery efforts. Different from the HDAC family, HDAC6 is constituted by a heterodimeric structure with two different catalytic domains, namely CD1 and CD2. Intriguingly, the specific biological relevance of each domain is not fully understood yet. However, a wealth of structural and biochemical data collected on HDAC, and in particular more recently on HDAC6, has revealed that the active sites in both CD1 and CD2 conserve a similar structural architecture. In the present work, we have performed a detailed investigation on the mechanistic aspects of catalysis of HDAC6, comparatively analyzing the enzymatic reaction mechanism in CD1 and CD2. Using density functional theory-based computations, we found that the rate-limiting step of the reaction is the nucleophilic attack of the catalytic water for peptide bond hydrolysis in both domains. Remarkably, the transition state for such a step presents a penta-coordinated metal center. In this regard, we have further validated the crucial contribution to catalysis of Tyr residues (Tyr363 in CD1 and Tyr745 in CD2) for the activation of the substrate, in line with kinetics measures. Importantly, we have quantified the effect on catalysis by the positively charged Lys330 residue in CD1. An analogous contribution is missing in CD2. Our calculations also evidence the contribution of distal residues from the reaction center, such as Gly201/582 and Phe202/583. Taken together, our quantum calculations dissect the catalytic mechanism in HDAC6. These findings may help future experimental studies and drug design efforts for selective HDAC6 targeting.

The Dawn of Metal-Oxo Clusters as Artificial Proteases: From Discovery to the Present and Beyond.

The selective cleavage of peptide bonds in proteins is of paramount importance in many areas of the biological and medical sciences, playing a key role in protein structure/function/folding analysis, protein engineering, and targeted proteolytic drug design. Current applications that depend on selective protein hydrolysis largely rely on costly proteases such as trypsin, which are sensitive to the pH, ionic strength, and temperature conditions. Moreover, >95% of peptides deposited in databases are generated from trypsin digests, restricting the information within the analyzed proteomes. On the other hand, harsh and toxic chemical reagents such as BrCN are very active but cause permanent modifications of certain amino acid residues. Consequently, transition-metal complexes have emerged as smooth and selective artificial proteases owing to their ability to provide larger fragments and complementary structural information. In the past decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC) and pioneered a distinctive approach to the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control the regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as the protein surface charge, metal coordination to specific side chains, and hydrogen bonding between the protein surface and the MOC scaffold.In this Account, we present a critical overview of our detailed kinetic, spectroscopic, and crystallographic studies in MOC-assisted peptide bond hydrolysis, from its origins to the current rational and detailed mechanistic understanding. To this end, reactivity trends related to the structure and properties of MOCs based on the hydrolysis of small model peptides and key structural aspects governing the selectivity of protein hydrolysis are presented. Finally, our endeavors in seeking the next generation of heterogeneous MOC-based proteases are briefly discussed by embedding MOCs in metal-organic frameworks or using them as discrete nanoclusters in the development of artificial protease-like materials (i.e., nanozymes). The deep and comprehensive understanding sought experimentally and theoretically over the years in aqueous systems with intrinsic polar and charged substrates provides a unique view of the reactivity between inorganic moieties and biomolecules, thereby broadly impacting several different fields (e.g., catalysis in biochemistry, inorganic chemistry, and organic chemistry).

The effect of pH and incubation time on rude protease enzymes activity of Bacillus mycoides from anchovy isolates (Stolephorus sp.)

Proteases are several enzymes that are in important positions in the enzyme industry. eyebrow hydrolysis of peptide bonds on protein. Bacillus mycoides bacteria can grow in Nutrient Broth medium, these bacteria are included in facultative anaerobic bacteria. Bacillus mycoides are resistant to penicillin, positive to form Beta-hemolysis, positively catalyze hydrogen without oxidase, positively reduce nitrates and reduce methylene This study aims to determine the effect of pH and time on the protease enzyme activity of Bacillus mycoides from salted anchovy isolates. This research was conducted using a descriptive method with the treatment of pH concentration which has 5 levels, namely pH 5, 6, 7, 8 and 9 which are grouped based on the incubation time which has 4 levels, namely 6 hours, 12 hours, 18 hours and 24 hours and each was repeated 2 times. The results showed that pH and time affected the protease enzyme activity of Bacillus mycoides from salted anchovy isolate. Different pH treatments gave significantly different effects (F count> F5%) and the optimum activity was obtained at pH 5 of 5.243 U/ml. The different incubation time treatments had a significantly different effect (F count> F5%) and the optimum activity at 24 hours was 5.154 U/ml.

Mathematical modeling of enzymatic hydrolysis of soybean meal protein concentrate

This study aimed to model the enzymatic hydrolysis of proteins present in soybean protein concentrate meal using two steps. In the first step, the concentration of hydrolyzed peptide bonds was described as a function of time, using two kinetic models. In the second step, the way the enzyme breaks the protein down into smaller parts was investigated and modeled using an artificial neural network. Enzymatic reaction assays used for modeling were performed at pH 8, 9, and 10. It was observed that although the pH influenced the behavior of the reaction rate and the time profile of the degree of hydrolysis (DH), the peptide profile was only a function of DH, and not explicitly a function of pH. These two steps provide a combined model that enables a more detailed understanding of the reaction, showing how it advances, and how product profile varies as the reaction progresses.

Peptide Hydrolysis by Metal (Oxa)cyclen Complexes: Revisiting the Mechanism and Assessing Ligand Effects.

The mechanism responsible for peptide bond hydrolysis by Co(III) and Cu(II) complexes with (oxa)cyclen ligands has been revisited by means of computational tools. We propose that the mechanism starts by substrate coordination and an outer-sphere attack on the amide C atom of a solvent water molecule assisted by the metal hydroxo moiety as a general base, which occurs through six-membered ring transition states. This new mechanism represents a more likely scenario than the previously proposed mechanisms that involved an inner-sphere nucleophilic attack through more strained four-membered rings transition states. The corresponding computed overall free-energy barrier of 25.2 kcal mol-1 for hydrolysis of the peptide bond in Phe-Ala by a cobalt(III) oxacyclen catalyst (1) is consistent with the experimental values obtained from rate constants. Also, we assessed the influence of the nature of the ligand throughout a systematic replacement of N by O atoms in the (oxa)cyclen ligand. Increasing the number of coordinating O atoms accelerates the reaction by increasing the Lewis acidity of the metal ion. On the other hand, the higher reactivity observed for the copper(II) oxacyclen catalyst with respect to the analogous Co(III) complex can be attributed to the larger Brönsted basicity of the copper(II) hydroxo ligand. Ultimately, the detailed understanding of the ligand and metal nature effects allowed us to identify the double role of the metal hydroxo complexes as Lewis acids and Brönsted bases and to rationalize the observed reactivity trends.

Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically β1, β2, and β5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits β1, β2, and β5 can be substituted by the immune βli, β2i, and β5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

Heterogeneous nanozymatic activity of Hf oxo-clusters embedded in a metal-organic framework towards peptide bond hydrolysis.

Materials with enzyme-like activities and proteolytic potential are emerging as a robust and effective alternative to natural enzymes. Herein, a Hf6O8-based NU-1000 metal organic framework (Hf-MOF) is shown to act as a heterogeneous catalyst for the hydrolysis of peptide bonds under mild conditions. In the presence of Hf-MOF, a glycylglycine model dipeptide was hydrolysed with a rate constant of kobs = 8.33 × 10-7 s-1 (half-life (t1/2) of 231 h) at 60 °C and pD 7.4, which is significantly faster than the uncatalyzed reaction. Other Gly-X peptides (X = Ser, Asp, Ile, Ala, and His) were also smoothly hydrolysed under the same conditions with similar rates, except for the faster reactions observed for Gly-His and Gly-Ser. Moreover, the Hf6O8-based NU-1000 MOF also exhibits a high selectivity in the cleavage of a protein substrate, hen egg white lysozyme (HEWL). Our results suggest that embedding Hf6O8 oxo-clusters is an efficient strategy to conserve the hydrolytic activity while smoothing the strong substrate adsorption previously observed for a discrete Hf oxo-cluster that hindered further development of its proteolytic potential. Furthermore, comparison with isostructural Zr-NU-1000 shows that although the Hf variant afforded the same cleavage pattern towards HEWL but slightly slower reaction rates, it exhibited a larger stability window and a better recyclability profile. The results suggest that these differences originate from the intrinsic differences between HfIV and ZrIV centers, and from the lower surface area of Hf-NU-1000 in comparison to Zr-NU-1000.

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

Proteomics approaches for the identification of protease substrates during virus infection.

Proteases precisely and irreversibly catalyze the hydrolysis of peptide bonds, regulating the fate, localization, and activity of many proteins. Consequently, proteolytic activity plays an important role in fundamental cellular processes such as differentiation and migration, immunological and inflammatory reactions, apoptosis and survival. During virus infection, host proteases are involved in several processes, from cell entry to initiation, progression and resolution of inflammation. On the other hand, many viruses encode their own highly specific proteases, responsible for the proteolytic processing of viral proteins, but, at the same time, to cleave host proteins to corrupt antiviral host responses and adjust protein activity to favor viral replication. Traditionally, protease substrate identification has been addressed by means of hypothesis-driven approaches, but recent advances in proteomics have made a toolkit available to uncover the extensive repertoire of host proteins cleaved during infection, either by viral or host proteases. Here, we review the currently available proteomics-based methods that can and have contributed to the systematic and unbiased identification of new protease substrates in the context of virus-host interactions. The role of specific proteases during the course of virus infections will also be highlighted.

Characteristics of matrix metalloproteinases and their role in embryogenesis of the mammalian respiratory system

Summary The human respiratory system appears as an outgrowth from the ventral wall of the primary foregut and its development includes a series of subsequent processes, dependent on the interactions between endothelial cells, respiratory epithelium and extracellular matrix (ECM). These interactions determine the acquisition of normal structural and functional features of the newly created tissues. The essential role in the morphogenesis of the respiratory system is performed by matrix metalloproteinases (MMPs). MMPs are endopeptidases containing zinc ion in their active center, necessary for the processes of hydrolysis of peptide bonds of substrates. The production of MMPs takes place in most connective tissue cells, leukocytes, macrophages, vascular endothelial cells as well as in neurons, glial cells and in tumor cells. Like other proteolytic enzymes, MMPs are produced and secreted in the form of inactive pro-enzymes, and their activation occurs in the extracellular space. MMPs perform both physiological and pathological functions during tissue modeling and their role in embryogenesis is based on the regulation of angiogenesis processes, stroma formation and cells migration. This article aims to characterize, discuss and demonstrate the activity and the role of MMPs in the subsequent stages of respiratory development.

Reversed Proteolysis—Proteases as Peptide Ligases

Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress.

Tryptophan Fluorescence and Time-Lag Hydrolysis of Peptide Bonds during Degradation of β-Lactoglobulin by Trypsin

The opening of protein globules and corresponding exposure of their internal peptide bonds, the so-called demasking effect, is required for successful hydrolysis of peptide bonds by proteases. Under the proteolytic action of trypsin on β-lactoglobulin (β-LG), the evolution of tryptophan fluorescence spectra showed that the demasking process consists of two stages with different demasking rate constants for each stage. It was found that the ratio of these constants depends on the concentration of trypsin and changes are approximately threefold when the concentration of trypsin changes in the range of 0.3–15 mg/L. Simulation of hydrolysis taking into account the demasking effect demonstrated how the apparent first-order rate constants obtained experimentally are related to the true hydrolysis rate constants and demasking parameters. The lag phase in the kinetic curves corresponding to the hydrolysis of various peptide bonds in β-LG was also analyzed. The increased lag times indicated sites that are hydrolyzed by a two-stage demasking mechanism.

Discrimination of enzyme–substrate complexes by reactivity using the electron density analysis: peptide bond hydrolysis by the matrix metalloproteinase-2

The results of quantum mechanics/molecular mechanics calculations of electron density changes upon oligopeptide hydrolysis by the matrix metalloproteinase-2 are utilized to discriminate between reactive and non-reactive enzyme–substrate complexes. Electron density depletion regions on the carbonyl carbon atom attacked by a catalytic water molecule are found on the 2D maps of electron density Laplacian in the reactive complexes. Also, the computed Fukui function quantitatively describes reactivity of the nucleophilic and electrophilic sites.