Alkyladenine DNA glycosylase (AAG) is a critical enzyme in the base excision repair (BER) pathway that safeguards genome integrity by removing structurally diverse alkylated and deaminated purine lesions from DNA. It serves as a primary defense against alkylation-induced mutations, which are linked to cancer development, chronic inflammation, and neurodegenerative diseases. Single nucleotide variants (SNVs) in the gene coding region have the potential to alter the enzyme's functionality, potentially modulating the repair capacity and affecting response and prognosis following chemoradiotherapy. In our study, we investigated three SNVs that lead to amino acid class changes in regions involved in DNA substrate coordination: R145H, G163S, and R197C using an in vitro approach. Using biochemical assays and molecular dynamics simulations, we evaluated the thermal stability, DNA binding affinity, and glycosylase activity of AAG variants toward hypoxanthine (Hx) and 1,N6-ethenoadenosine (εA) containing substrates. The G163S variant showed reduced thermal stability due to the conformational strain in the β-hairpin loop that intercalates in DNA, but retained εA excision activity comparable to that of wild-type AAG, while losing activity against Hx-containing DNA. The R197C variant had a four-fold reduction in DNA binding affinity for both substrates, and was catalytically inactive, unable to excise either damaged bases. This loss of function correlated with the rearrangement of the 201-210 loop and the reorientation of Arg-201 and Arg-207, which disrupts critical DNA contacts. However, the R145H variant retained near-wild-type thermal stability and activity on both substrates, despite bioinformatic predictions of deleterious effect. Molecular dynamics simulations revealed variant-specific structural disruptions. The data obtained underscore the importance of experimental validation in assessing the functional impact SNVs.
The heat shock protein 70 (HSP70)-HSP90 organizing protein (HOP), also known as STIP1, is a vital co-chaperone that mediates the transfer of oncogenic client proteins between HSP70 and HSP90. While HOP's canonical role as a co-chaperone involves coordinating HSP70 and HSP90 activity to facilitate the folding, maturation, and stabilization of selected oncogenic client proteins, the interaction between HOP and the cellular prion protein (PrPC) has recently emerged as a significant driver of tumor progression, metastasis, and the maintenance of cancer stem cell characteristics. This study utilizes an integrated computational pipeline, including molecular docking and extensive molecular dynamics (MD) simulations with a cumulative sampling time approaching 1.5 μs, to provide the first comprehensive structural roadmap of the human HOP-PrPC interaction. Our results elucidate a hierarchical binding mechanism where the PrPC protein initially targets the TPR2A domain of HOP with a binding affinity of -88.01 ± 10.88 kcal/mol, subsequently recruiting the TPR2B and DP2 modules to achieve a high-affinity "locked" state of -152.38 ± 29.79 kcal/mol. Critical interface hotspots were identified at HOP residues Leu187 and Pro211 via saturation mutagenesis. Furthermore, we evaluated the inhibitory potential of repurposed small molecules and the amphipathic peptide Melittin. While the FDA-approved drug Tarceva (Erlotinib) restricts PrP engagement to the TPR2A domain by inducing structural compaction in HOP, Melittin emerged as a superior inhibitor. Melittin binding at the TPR1-TPR2A interface triggers the complete physical detachment of the PrPC peptide. Furthermore, against the full-length PrPC protein, Melittin dismantling the multi-domain 'locked' state and causes a drastic reduction in binding affinity, restricting the protein almost entirely to the TPR2A domain. These findings demonstrate that the conformational plasticity of HOP can be strategically exploited for targeted protein-protein interaction (PPI) disruption in cancer therapy.
Receptor tyrosine kinases (RTKs) are increasingly understood to signal beyond the plasma membrane. However, the role of Discoidin domain receptor 2 (DDR2), which is activated by collagen, in neurodegeneration remains poorly understood. This study uses immunoprecipitation followed by mass spectrometry on cytoplasmic and nuclear fractions from a neuronal cell model to show that Aβ42 exposure is associated with a compartment-specific reorganization of DDR2-associated protein complexes, characterized by increased nuclear representation. This reveals a unique nuclear DDR2 interactome under amyloidogenic conditions. By exploring this non-canonical, compartment-specific DDR2 signaling pathway, our results offer new insights into how receptor signaling networks may be altered in Alzheimer's disease pathology. Additionally, we identify DDR2-related nuclear interactions as potential sites of dysregulation in neurodegeneration.
Proteins operate under competing demands imposed by stability, dynamics, and function, all of which are shaped by evolution. Local energetic frustration provides a quantitative framework to describe how these competing requirements are distributed within the native states of proteins, identifying regions where interactions are optimized and others where energetic conflicts are retained to enable functional behavior. In recent years, the study of local frustration has expanded significantly, driven by the integration of large-scale structural datasets and advances in artificial intelligence methods. Comparative analyses have shown that frustration patterns encode evolutionary pressures across protein families, with minimally frustrated interactions stabilizing structural cores and highly frustrated regions often associated with catalysis, binding, conformational transitions as well as pathogenic phenotypes. At the same time, modern protein language models and structure prediction methods seem to implicitly capture the statistical and structural features underlying frustration, enabling its prediction directly from sequence or structure at proteome scale. These developments suggest that local energetic frustration may be interpreted as an emergent property of the evolutionary information learned by AI models. Here, we review recent advances in the analysis and prediction of local frustration and discuss how this framework could provide mechanistic insights into protein evolution, conformational dynamics, and design. We further argue that incorporating frustration into computational and experimental strategies will be essential to move beyond purely stability-driven approaches toward the rational engineering of functional proteins.
The mammalian brain's functional complexity arises from the sophisticated architecture of neurons and glia. This network is essentially defined by its dynamic proteome, which reveals the functional execution underlying neural computation and disease. This review integrates the technological leap in neuroproteomics. It has moved beyond bulk tissue proteome cataloguing to high-sensitivity single-cell and spatial resolution. We detail how next-generation platforms, such as TIMS-PASEF and Orbitrap-Astral, have enabled deeper and faster phenotypic profiling of limited brain samples. However, the proteome coverage remains constrained by dynamic range, sample loss, ionisation bias and incomplete detection of low-abundance regulatory proteins. We further examine how such studies have revealed the proteomic remodelling that drives lineage specification and synaptic plasticity by linking temporal protein expression waves to biological function. Crucially, we delineate the clinical translational trajectory, illustrating how aberrant signatures are verified in cerebrospinal fluid (CSF) and validated in plasma to support precision medicine. Finally, we argue for the necessity of "fused" multi-omics integration and Artificial Intelligence (AI) to decode the non-linear molecular logic of brain pathology.
Crosstalk among tRNA modification proteins have been implicated in important functional roles in biology. We previously reported that Physcomitrium tRNA (cytosine38-C5)-methyltransferase, TRDMT1/DNMT2 catalyses C38, C48 and C49 methylation in tRNAAsp(GUC) and plays a crucial role in regulating transcription/stability of tRNAAsp(GUC) under oxidative stress. To gain insight into its precise mode of regulation, in the present study we show that moss TRDMT1/DNMT2 participates in broader networks of tRNA modification pathways. Using in silico methods we first identified that the yeast homologs of m7G46 methyltransferase Trm8 that catalyses m7G46 methylation, its obligate partner protein Trm82 and the Queuine tRNA ribosyl transferase (QTRT1) exist in a functional network with PpTRDMT1/PpDNMT2. To examine genetic interaction between PpTRDMT1/PpDNMT2, Trm8-82 and QTRT1, PpTRDMT1/PpDNMT2 loss-of-function mutants (ppdnmt2) and ppdnmt2 rescued lines (PpDNMT2-c) were used and transcript abundance of each gene was examined by qRT-PCR in background of these plants. Thereafter, physical interaction between PpTrm8/8 L1/8 L2 and PpQTRT1 with specific catalytic motifs in PpTRDMT1/PpDNMT2 were studied by yeast two-hybrid assay. The observed protein-protein interaction was also supported by in silico analysis of docked complexes of stretches of PpTRDMT1/PpDNMT2 and the Trm8 homologs. Alanine scanning mutagenesis study identified Threonine 11 in PpTRDMT1/PpDNMT2 located close to catalytic motif IV to be crucial for stable complex formation between PpTRDMT1/PpDNMT2 and its binding partners. On the basis of the results obtained we propose that pathways leading to m7G46 modification by tRNA-Guanine-N-7-methyltransferases and incorporation of Queuine by QTRT1 may be modulated at transcriptional/post-transcriptional levels by PpTRDMT1/PpDNMT2 function in P. patens. SIGNIFICANCE STATEMENT: tRNA modifying enzymes form a network in the moss P. patens.
The pathological accumulation of α-synuclein (α-syn) into amyloid fibrils is a key hallmark of Parkinson's disease and related synucleinopathies. Inhibiting the early stages of α-syn aggregation remains a major therapeutic challenge. In this work, we report the fabrication and investigation of five sugar-based nanoparticles (NPs), including glucose, fructose, maltose, sucrose, and trehalose, and their ability to inhibit α-synuclein aggregation. Using a combination of biophysical strategies, comprising thioflavin-T fluorescence, dynamic light scattering, circular dichroism, and confocal microscopy, we illustrate that sugar NPs prevent β-sheet formation and the growth of α-syn fibrils in a concentration-dependent manner. Isothermal titration calorimetry revealed spontaneous, high-affinity interactions between α-syn and sugar NPs, suggesting direct binding to aggregation-prone regions. Importantly, cytotoxicity assays using SH-SY5Y neuroblastoma cells showed that NP-treated α-syn aggregates exhibited significantly reduced neurotoxicity. Collectively, these findings demonstrate that sugar-derived nanoparticles act as potent inhibitors of α-syn aggregation during the lag phase, thereby stabilizing non-toxic conformers. These results highlight the promise of sugar-based nanostructures as biocompatible and mechanistically active molecules for modulating synucleopathies associated with various neurodegenerative disorders.
Manganese superoxide dismutase (MnSOD), localized in the mitochondrial matrix, is considered pivotal enzyme in the mitochondrial powerhouse. This is primarily due to its central role in the scavenging of excess superoxide anions (O2•-) produced during the electron transport chain. In this study, a highly thermostable MnSOD from Lantana camara (LcMnSOD) was identified via SOD activity assays. Isozyme profiling and thermostability assays demonstrated a high thermal stability of LcMnSOD as it was found to be active even at the temperature exceeding 80 °C. Full-length cDNA (944 bp) encoding LcMnSOD (675 bp) was amplified by rapid amplification of cDNA ends (RACE), followed by recombinant expression in Escherichia coli and further purification via affinity chromatography. The purified enzyme (∼24 kDa) exhibited Km value of 0.026 ± 0.001 μM, Vmax value of 266.29 ± 10.42 Units/mg and was functional across a broad pH (5.0-9.0) and temperature (4-70 °C) range. LcMnSOD existed as a homotetramer and had a high α-helical content, as confirmed by in silico and circular dichroism (CD) analysis. Interestingly, LcMnSOD was resistant to heat inactivation at 80 °C (kd = 0.0063 ± 0.0004 min-1, t1/2 = 116.48 ± 13.96 min), and was also stable to varying concentrations of denaturants, inhibitors, and reducing agents. LcMnSOD could maintain its activity at high temperatures by maintaining a dense hydrogen-bonding network, a robust hydrophobic core, and structural integrity of its catalytic site, ensuring accessibility of substrate-binding residues, as revealed by Molecular dynamics (MD) simulation studies. Owing to its remarkable stability and functional robustness, LcMnSOD holds promise for biotechnological applications.
The global rise of drug-resistant Mycobacterium tuberculosis (Mtb) underscores an urgent need for antitubercular agents with novel targets and mechanisms of action. Among these, the de novo purine biosynthesis pathway is essential for Mtb growth and survival, making its constituent enzymes attractive targets for therapeutic intervention. Within this pathway, adenylosuccinate (ADS) synthetase (ADSS) Rv0357c catalyzes the first committed step in biosynthesis of adenosine monophosphate (AMP) by converting inosine monophosphate (IMP) to ADS through a GTP-dependent reaction with l-aspartate. Despite its importance, Mtb ADSS remains poorly characterized at the biochemical level. In this study, we report the expression, purification, and enzymatic characterization of recombinant Mtb ADSS. To overcome the challenge of the enzyme being predominantly expressed as inclusion bodies in Escherichia coli, we established both protein refolding and chaperone-assisted expression strategies to obtain soluble, catalytically active protein. Using complementary spectrophotometric, colorimetric, and fluorescence-based assays, we determined steady-state kinetic parameters and confirmed robust ADSS activity consistent with Michaelis-Menten behaviour. Furthermore, we developed scalable, nonradioactive assays compatible with high-throughput screening (HTS), enabling the quantitative monitoring of ADSS activity via GTP hydrolysis and phosphate release. As a proof of concept, the MESG assay successfully detected inhibition of Mtb ADSS by the previously reported ADSS inhibitor Aurodox, demonstrating its utility for inhibitor characterization and screening. Collectively, these results provide the first comprehensive biochemical framework for studying Mtb ADSS and establish a foundation for structure-guided inhibitor discovery targeting purine biosynthesis as a novel antitubercular strategy.
Catharanthus roseus, a medicinal plant known for its anti-cancer monoterpene indole alkaloids vinblastine and vincristine, possesses two distinct types of geranyl pyrophosphate synthases (GPPS). A homomeric GPPS synthesizes geranyl pyrophosphate (GPP) as the sole product in the mitochondria, while a heteromeric GPPS comprising of a large subunit (LSU) and small subunit (SSU) is responsible for the GPP production in the chloroplasts. In the present study, in silico structural characterization and structure-activity correlation analysis of large subunit of the heteromeric GPPS (CrGPPS.LSU, GenBank Accession: JX417183) of C. roseus has been carried out. CrGPPS.LSU belongs to the type II trans-prenyltransferase family, having seven conserved regions. The protein comprises of 383 amino acids, with the first 63 amino acids belonging to the chloroplast transfer signal peptide sequence. CrGPPS.LSU showed the highest sequence identity with the heterotetrameric geranyl pyrophosphate synthase from Mentha piperita. A 3 dimensional (3D) structural model of the protein was obtained using AlphaFold 3, and was compared with other structurally characterized GPPS/GGPPS from plants, which revealed specific amino acid residues responsible for product formation in CrGPPS.LSU. The homomeric association of CrGPPS.LSU may be mediated through amino acid residues H109, D141, E149 and K172, while the presence of F148 might restrict the product chain elongation. An in silico mutation analysis of selected amino acids of CrGPPS.LSU further confirmed that the heterodimeric association of CrGPPS.LSU with a Type-I small subunit (CrGPPS.SSU; GenBank Accession: JX417184) is stabilized through interactions involving amino acid residues H109, D141, E133, D134, E149 and E177.
Allantoin plays a major role in nitrogen mobilization as well as abiotic stress tolerance in plants, and uricase enzyme is the key rate-limiting enzyme for the biosynthesis of allantoin in peroxisomes. However, the molecular mechanisms underlying stress-induced allantoin accumulation as well as enhanced uricase activity, along with the evolutionary conservation, structural diversification and regulatory characteristics of plant uricase enzymes, remain poorly understood. This study investigates evolutionary relationships, functional variations and catalytic mechanisms of the uricase enzymes among 157 plant species. Phylogenetic analysis grouped these species into three major clades, indicating a progression from thallophytes to higher plants. Motif analysis revealed the presences of a conserved Pfam01014 (uricase) domain across the plant species. Uricase activity analysis of 22 representative species showed the highest activity in ureidic legumes, including Vigna radiata and Glycine max, and the lowest in non-leguminous species such as Cucumis sativus. Moreover, promoter analysis of the uricase gene identified diverse cis-regulatory elements associated with stress, hormone responses and plant development. Structural modelling, docking and sequence alignments revealed conserved substrate-binding residues. Biophysical characterization of recombinantly purified uricase from Oryza coarctata using fluorescence spectroscopy demonstrated uric acid binding with a dissociation constant (KD) of 10.89 μM, positive cooperativity and the Michaelis constant (KM) of 23.96 μM. Circular dichroism showed a conformational shift from α-helix to β-sheet upon ligand binding and molecular dynamics simulations supported stable urate interactions in O. coarctata. This work highlights the evolutionary conservation and functional divergence of uricase, featuring unique catalytic adaptations in ureidic legumes.
Arginylation is a well-known, evolutionarily conserved, and recognizable post-translational modification (PTM). In this modification, an arginine residue is enzymatically attached to the target residue, such as aspartic acid (D), glutamic acid (E), and oxidized cysteine (Cys-SO₂H, Cys-SO₃H) by arginyltransferase 1 (ATE1) utilizing arginyl-tRNA. This phenomenon occurs both at the N-terminal and mid-chain of a protein. Arginylation is required for various biological activities like overall cellular development, cytoskeletal organization, and cellular stress adaptation. For predicting arginylation likelihood, only laboratory-based experimental procedures are currently available. To solve this, we developed the web server ARGpredict to predict arginylation events in protein sequences. The designed web server analyzes the arginylation target sites and assigns specific scores. To evaluate its effectiveness, this web server was tested against various known and experimentally validated datasets. ARGpredict yielded promising prediction results, which were also verified by statistical methods. The web server is available at https://iitg.ac.in/sachinku/ARGpredict/.
In this study, we developed and applied an integrative computational workflow for the systematic identification and prioritization of candidate allosteric pockets across all four glycolytic enzymes: three from Staphylococcus aureus-phosphoglucose isomerase (PGI), phosphoglycerate kinase (PGK), and enolase- and one representative hexokinase from Plasmodium vivax, included due to the absence of an experimentally determined three-dimensional structure for the S. aureus ortholog. Solvent mapping using FTMap and FTMove across oligomeric ensembles revealed multiple high-confidence cavities predominantly located at subunit interfaces, in addition to canonical catalytic sites. Independent evaluation with CavityPlus supported the presence and druggability of these pockets. Hexokinase presented 12 interface-associated pockets that emerged only upon oligomer formation and remained stable across 300 FTMove-derived conformers. PGI and PGK displayed interface- and hinge-associated cavities linked to known global motions, while the octameric enolase showed prominent central and peripheral inter-dimer pockets. Candidate pockets were subsequently evaluated using CorrSite, ESSA, PASSer, and AlloSigMA to assess features associated with allosteric communication, energetic coupling, and protein dynamics. High-confidence candidate sites were prioritized based on consensus across these complementary computational approaches. Across all four enzymes, interface-localized pockets consistently emerged as promising candidate regulatory regions, suggesting that protein-protein interfaces may represent valuable targets for allosteric modulation. Several predicted pockets, particularly in PGI and enolase, exhibited low sequence and structural similarity to the corresponding human homologs, indicating their potential for selective inhibitor design. Overall, this integrated computational framework provides a systematic strategy for identifying and prioritizing candidate allosteric pockets for future structural, biochemical, and structure-based drug discovery.
The luciferase from the mesopelagic planktonic crustacean Gaussia princeps has garnered attention as reporter protein due to its small size and its ability to produce a very bright luminescence. Over the past two decades much research has focused on the development of improved mutant variants as well as variants designed by combining sequence information from luciferases from several related copepod species.Gaussia luciferase is however of basic scientific interest because it is unusual in two main respects. First, structural analysis of Gaussia luciferase has demonstrated that this enzyme is extensively disordered with little hydrophobic core. This has complicated the identification of a substrate binding site and elucidation of the enzymatic mechanism remains obscure. Second, this luciferase is subject to rapid irreversible inactivation upon oxidation of its substrate, coelenterazine. These findings open fundamental enzymology questions and have significant implications for the use of Gaussia luciferase in life sciences; areas such as bioluminescence-based reporting assays and in high-throughput screening. Thus, past research is examined considering these recent findings, and with special emphasis on their implications for consistent assays and evaluation of mutant variants.
Adenosine triphosphate phosphoribosyltransferase (ATP-PRT), a member of the phosphoribosyltransferase (PRT) superfamily of enzymes, catalyses the first step in the histidine biosynthesis pathway, which involves the nucleophilic substitution of ATP onto phosphoribosyl pyrophosphate (PRPP) to generate phosphoribosyl-ATP (PR-ATP) and pyrophosphate (PPi). The three-dimensional structure of short-form ATP-PRT from Acinetobacter baumannii (AbHisGS) was determined using the X-ray crystallographic method. The structure determination revealed two crystallographically independent molecules in the asymmetric unit that formed an antiparallel homodimer. In all the structures of HisGS proteins reported so far, the N-terminal segment is either absent from the amino acid sequence or not observed in the structure. This is the first structure where the N-terminal segment has been clearly observed. However, the most remarkable observation pertains to observing a Mg2+ ion in the structure with the help of the N-terminal segment, which is loosely held with six coordination linkages, out of which one contact is made to the carbonyl oxygen atom of Asn16 at an average distance of 2.06 Å, and the other five contacts were with water oxygen atoms at distances varying from 2.40 to 2.59 Å. All the coordinating water oxygen atoms were held in place by multiple hydrogen bonds from surrounding protein atoms. The relatively loose coordination environment of the Mg2+ ion indicates temporary sequestration of Mg2+, which may be released during catalytic need.
Collagen is a highly abundant extracellular matrix protein characterized by repetitive Gly-X-Y motifs and extensive post-translational modifications (PTMs), including hydroxylation of proline and lysine residues as well as O-glycosylation of hydroxylysine residues. This PTM density generates numerous peptide isoforms that challenge confident identification and quantification by LC-MS/MS. In this study, we have performed a systematic analysis of co-eluted, near-isobaric collagen peptides arising from site-specific PTM heterogeneity from the ECM of different mouse tissues. Using a publicly available healthy mouse tissue ECM proteomics dataset and a PTM-aware database search strategy, we demonstrate that near-isobaric collagen peptides frequently co-elute and co-fragment, producing chimeric spectra with high-confidence peptide-spectrum matches for multiple isoforms. Despite robust MS/MS evidence, overlapping retention times (RT) and identical m/z values prevent unambiguous localization of modification sites, necessitating exclusion from quantitative analyses. Our findings show that such co-elution reflects genuine biological heterogeneity rather than technical noise and highlight a critical gap between the complexity of collagen PTMs and current computational proteomics workflows, underscoring the need for advanced separation and PTM-aware analytical strategies.
The Janus kinase (JAK)/STAT signaling pathway plays a pivotal role in cancer biology as well as in inflammatory and autoimmune disorders such as psoriasis. Recent advances in biomedical research and targeted therapies have highlighted the importance of computational approaches for accelerating the discovery of selective kinase inhibitors. This study aimed to develop a robust computational framework for predicting the inhibitory potency of JAK2 ligands and for analyzing their binding interactions using structure-based methods. A curated dataset of 1869 chemically valid JAK2 ligands with experimentally reported Ki values was compiled, standardized, and converted to pKi. Using this dataset, a bond-aware graph neural network (GNN) was trained and evaluated for pKi prediction. Top-ranked predicted ligands were further examined via molecular docking, pharmacophore modeling, and molecular dynamics simulations to assess their interactions within the JAK2 ATP-binding site. The proposed model achieved strong predictive performance, yielding an average test-set R2 of 0.91 ± 0.01, MAE of 0.14 ± 0.01, and RMSE of 0.26 ± 0.02 across repeated data splits. Structure-based analyses supported the predicted binding poses and identified key stabilizing interactions within the JAK2 ATP-binding site. Overall, this integrative computational framework provides a reliable approach for predicting JAK2 inhibitory potency and offers mechanistic insights that may support the computational prioritization of candidate molecules for future experimental evaluation.
Serratiopeptidase is a metalloprotease enzyme mainly used for its anti-inflammatory and therapeutic applications. The functional performance of serratiopeptidase is compromised by the reduced enzymatic activity at extreme pH levels especially in oral administration. To address the problem, the present research employed the structure-based rational design strategy to assess the possibility of modifying the functional performance of serratiopeptidase by introducing a point mutation, N412D, which might improve the functional performance of the metalloprotease under extreme pH levels. Constant pH molecular dynamics simulations were used as an in-silico tool for the qualitative analysis of the wild type (WT) and the mutated serratiopeptidase under variable pH environments. To perform the functional evaluation, the mutated serratiopeptidase was expressed, refolded, and purified using the Escherichia coli expression host. Retained caseinolytic activity was analyzed at different pH levels ranging from 3.0 to 11.0 to evaluate the functional property of serratiopeptidase. It has been noted that the functional activity of mutated serratiopeptidase is slightly changed with respect to an optimal pH, coupled with high retained caseinolytic activity at variable pH levels. The highest improvement in the retained caseinolytic activity was observed under alkaline pH. A small improvement in the retained caseinolytic activity was also observed under acidic pH levels. This research demonstrated the importance of rational design strategies in the selection of the mutation site which might influence the functional resilience of serratiopeptidase under different pH levels.
The existence of multiple molecular forms of enzymes, genetic polymorphism and functional promiscuity raise the question of the identity of active center(s) responsible for several activities. In the present review, we recapitulate the general strategy, implementing the simple and rigorous inhibition kinetic method for probing the existence of single or multiple active sites on enzyme molecules. The model enzyme we chose to illustrate this approach is human butyrylcholinesterase, an enzyme that shows a complex functional (promiscuity), structural (multiple oligomeric forms) and genetic polymorphisms (numerous allelozymes and isoenzymes).This classical active site discrimination method is based on the analysis of enzyme irreversible inhibition profiles of enzymes under first-order conditions by monitoring the progressive enzyme activity decay with two reporter substrates of different specificity. The use of chiral irreversible inhibitors and/or chiral reporter substrates provides additional kinetic information about preferential enantioselectivity or binding complementarity of the target enzyme, allowing selection of the best inhibitors or substrates. Then, additional investigations, using structural methods (X-ray structure analysis, mass spectrometry), in silico simulations and classical biochemical methods (electrophoresis, PCR) provide definitive answers.
