Pseudomonas aeruginosa infection. This study aimed to design compounds with a new mechanism through competitive inhibitory activity against phosphomannomutase/phosphoglucomutase (PMM/PGM), using in vitro assessment and a computational (in silico) approach. The active site of PMM/PGM was assessed through molecular redocking using L-tartaric acid as the native ligand and other small molecules, such as glucaric acid, D-sorbitol, and ascorbic acid. The docking program set the small molecules to the active site, showing a stable complex formation. Analysis of structural similarity, bioavailability, absorption, distribution, metabolism, excretion, and toxicity properties proved the potential application of ligands as an anti-biofilm. In vitro assessment with crystal violet showed that the ligands could reach up to 95.87% inhibition at different concentrations. The nitrocellulose membrane and scanning electron microscopic visualization showed that the untreated P. aeruginosa biofilm was denser than the ligand-treated biofilm.An anti-biofilm that can inhibit the matrix of biofilm formation is necessary to prevent recurrent and chronic  Pseudomonas aeruginosa (P. aeruginosa) is an environmental, opportunistic bacterium associated with various diseases with various clinical spectrums. It primarily causes ventilator-associated pneumonia (VAP) with high mortality/morbidity rates , D-sorbitol [DS], and ascorbic acid [AA] via ligand exploration based on the established criteria. These criteria include nontoxic, easy to obtain and reported safety for use .5…(Arg11)N-HH2 at 1.68 Å, [5…(Arg11)N-HH12 at 2.26 Å, [5…(Arg16)N-HH12 at 1.71 Å, [6…(His105)N-HE2 at 3.05 Å, [7…(Arg243)N-HE at 1.80 Å, [4…(His304)N-HE2 at 2.90 Å, and [4-H…(His325)NE2 at 2.07 Å), DS-PMM/PGM ([6…(Arg11)N-HH22 at 1.74 Å, [3…(His304)N-HE2 at 2.07 Å, and [4…(His304)N-HE2 at 2.75 Å), and AA-PMM/PGM ([16(O5)…(His105)ND1 at 2.59 Å and [8…(Lys114)N-HZ2 at 1.99 Å).The grid score of each ligand was determined . This score is associated with the binding affinity . The higher the negative grid score, the more vital the ligand interaction on the receptor’s active site from a thermodynamic aspect. The ligands and receptor showed conformation fitness, interacting via several receptor residues on the receptor’s active site, including GA-PMM/PGM , DS-PMM/PGM , and AA-PMM/PGM . In particular, the hydrogen bond interaction with each ligand plays an important role in the target protein inhibition mechanism because the H-bond parameter is a strong category in ligand–receptor interactions ,21. The H-bond interactions were further visualized in the form of a hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD). As shown in Data on the prediction of drug-likeness of each ligand molecule with a competitive inhibitory activity against PMM/PGM based on docking analysis are shown in 2 < TPSA < 130 Å2), insolubility (0 < ESOL < 6), saturation (0.25 < Csp3 < 1), and flexibility (0 < rot bonds < 9) [nds < 9) ,24. FurtMeanwhile, the predicting metabolic parameters showed that the antibiofilm candidates did not inhibit cytochrome isoenzymes (CYP), including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4. The absence of these isoenzymes indicated that each antibiofilm candidate was a promising drug candidate because it did not interfere with the CYP activity. Therefore, the antibiofilm candidate will not induce unwanted side effects on metabolic processes. In addition, the prediction of excretion showed that antibiofilm candidates were included in renal organic cation transporter-2 (OCT2) prediction, a subcategory of the non-substrate.Analysis of toxicity showed remarkable results, where all antibiofilm candidates showed non-toxicity and met the following criteria: non-AMES toxic, non-hepatotoxicity, and non-skin sensitization . Thus, o3 µg/mL. By contrast, GA or calcium D-saccharate tetrahydrate had >50% inhibitory biofilm formation activity at 2.5 × 102 µg/mL.The activity of each ligand as an anti-biofilm was evaluated at several concentration ranges using crystal violet assay. Based on the data presented in 5 µg/mL. AA had a >50% inhibitory activity against P. aeruginosa biofilm formation at 1.25 × 104 µg/mL, and its inhibitory activity reached 91.5% .P. aeruginosa biofilm comprised bacterial cells with some exopolysaccharides (EPSs). The bacterial cell walls appeared intact and closed together, attached, and covered by an EPS matrix. P. aeruginosa, which is commonly associated with pneumonia, interacts and interplays with other microbes in polymicrobial biofilms, thereby enhancing pathogenesis and affecting antimicrobial therapy. Therefore, the high mortality/morbidity rate associated with pneumonia and the increased antibiotic resistance worldwide have prompted studies on novel therapeutic strategies to combat polymicrobial infection in pneumonia. Rodrigues et al., (2017) proposed combinational antimicrobial therapy using one antibiotic  and anti-fungal  agent against the polymicrobial biofilms of P. aeruginosa and Candida albicans [P. aeruginosa and C. albicans. In addition, we evaluated the performance of several anti-biofilms of C. albicans that enhance the anti-fungal action of fluconazole [Rodrigues et al., 2017) showed the characteristics of polymicrobial biofilms associated with pneumonia, with bacterial–fungal polymicrobial biofilms colonizing the endotracheal surface. 017 showeconazole .P. aeruginosa anti-biofilms based on the competitive inhibitors of the PMM/PGM enzyme, which is responsible for extracellular matrix biosynthesis, was assessed. In the present study, substances with a structure similar to that of the PMM/PGM active site were identified, which served as ligands for this enzyme and a macromolecule. The ligands included the stereoisomer of the native substrate of PMM/PGM, mannose, and glucose and the ligand of S108A PMM/PGM (TLA). The results showed that three substances  matched the criteria. Based on the structure similarity between these ligands and TLA , the ligands should occupy the active site of PMM/PGM. Thus, these ligands may induce activity as PMM/PGM competitive inhibitors, which can be predicted via in silico analysis, thereby indicating its activity through in vitro assay.The exploration of several Theoretical studies on the use of substrate analogs as PMM/PGM inhibitors based on their structural similarity (mimic) to native ligands and their commercial availability can provide an efficient approach to drug design. In addition, the mechanism of PMM/PGM competitive inhibition was analyzed on the basis of the interaction of the inhibitor with residues on the receptor’s active site at a molecular level. Redocking determined the active site of PMM/PGM based on its interaction with TLA as the native ligand. Moreover, sphere cluster selection using Dock6 can facilitate easier identification of native ligand coordinates at the receptor active site ,28.Docking of the PMM/PGM ligand was conducted at the initial coordinates of the redocking results A. In addThe performance and characteristic prediction of competitive inhibitors were investigated via in silico pharmacokinetic studies. Furthermore, the pharmacokinetics of inhibitors were evaluated via several parameters, including drug-likeness, bioavailability, and ADMET. These parameters can provide information on biological activities based on their structure. For example, drug-likeness analysis observes drug permeability by considering Lipinski’s and Veber’s rules. A drug candidate has good permeability if it does not deviate by more than one Lipinski’s and Veber’s rule .P. aeruginosa. Then, this prediction has been proven via quantitative and qualitative in vitro assays.Previous pharmacokinetic studies confirmed that each inhibitor meets the criteria of oral drugs, showing that the development of an excellent oral drug had ≤2 deviations . TherefoP. aeruginosa biofilms. Using spectrophotometric analysis and the crystal violet method, biofilm formation was determined on the basis of optical density and [The first in vitro assay using the crystal violet method confirmed that the four ligand compounds could inhibit sity and ,31. In v3 µg/mL inhibits biofilm formation by almost 100%. This result indicates the application potential of TLA as an anti-biofilm candidate against P. aeruginosa biofilm. Previous studies reveal that tartaric acid and its derivates have antimicrobial activity against several bacteria [Tartaric acid derived from ascorbic acid as the native ligand represents aldaric acid, which can be found in different plants. Tartaric acid and its salts  are widely used in the food industry as flavoring agents, food additives, emulsifiers, antioxidant synergists, and sequestrates, which are safe for humans ,33. Crysbacteria ,35, but 2 µg/mL. GA in this assay has shown an inhibitory activity of >50% when the concentration is below 5 × 102 µg/mL.Walaszek (1997) reported that GA has anticancer and antioxidant properties. However, its anti-biofilm and antibacterial activities have not been evaluated. Calcium D-glucarate is a supplement for cancer prevention, liver detoxification, and hormone regulation. Moreover, it is a calcium salt of D-GA, a non-toxic compound found in several fruits and vegetables, particularly grapefruit, apples, oranges, and broccoli ,37. The S. mutant bacteria [P. aeruginosa biofilm formation at 4 × 105 µg/mL. Sorbitol is sugar alcohol with a sweet taste which is slowly digested in humans. This compound can be produced by reducing glucose, which converts the aldehyde group (-CHO) into primary alcohol or the hydroxyl group (-C(OH)H2). Sorbitol is widely found in fruits, and it is used in industries as an additive to food, cosmetics, and medicine [Sorbitol, a polyol group, inhibited biofilm formation in bacteria . The polmedicine .C. perfringens. Meanwhile, Serry et al., (2008) showed that AA served as an efflux pump inhibitor. Finally, Abbas et al., (2012) showed that sodium ascorbate inhibited planktonic growth in P. aeruginosa at 5–20 mg/mL.The inhibitory ability of AA describes the mechanism of PMM/PGM inhibition via substrate analogs. Several studies have shown the capability of AA to interfere with the physiology of microorganisms. For example, Novak and Fratamico (2004) reported the capability of AA to inhibit quorum sensing in P. aeruginosa biofilms [P. aeruginosa biofilm was denser than that of the ligand-treated biofilm. Based on this qualitative experiment, each test ligand reduced the formation of P. aeruginosa biofilms. More detailed evaluations of microscopic structures were conducted by scanning electron microscopy (SEM).The second in vitro assay used the colony biofilm method. This method is suitable for assessing the strength of an anti-biofilm or resistance to antibiotics. The nitrocellulose membrane is a good surface for growing biofilms . BiofilmP. aeruginosa on glass coverslips was monitored via SEM. This section showed that the cell morphology has changed with ligand treatment. The preparation of SEM comprises several steps that can prevent changes in the morphological structure, although the cell undergoes death [The third in vitro assay was performed using an electron microscope. es death .P. aeruginosa biofilms (control) comprise bacterial cells and an EPS matrix. P. aeruginosa biofilms grown in vitro are formed through several stages. First, planktonic bacteria attach to abiotic or biotic surfaces through flagella and adhesion. Second, an irreversible attachment will occur with the help of the SadB protein. Third, microcolonies are formed in the complex layer of biomolecules and the EPS matrix. Fourth, the biofilm matures. Fifth, bacteria are released from the biofilm to form a new biofilm [SEM analysis revealed that untreated  biofilm .3 µg/mL showed sparse cells without EPS, which is similar to the addition of GA (5 × 102 µg/mL), DS (4 × 105 µg/mL), and AA (5 × 104 µg/mL). The appearance of the bacterial cell wall in each treatment varied. The TLA and GA-treated cells and the cell walls of some bacteria were not intact and lysed. Meanwhile, the DS-treated cells were affected by osmotic pressure, thereby causing the cells to become turgid. Meanwhile, the cell walls remained intact. However, EPS was not found in AA-treated cells.All tested ligands were added after the first stage when bacterial cells were attached to the coverslips’ surface. Based on the stages of biofilm formation , the EPSP. aeruginosa was isolated from the sputum of patients with pneumonia. It was identified using VITEK 2 COMPACT  in Clinical Microbiology Laboratory DR. SOETOMO Hospital Surabaya (Lab. Number 10895) and was assayed to identify its biofilm formation capability. This isolate was stored at −80 °C in Tryptone Soya Broth (TSB) CMO129  supplemented with glycerol 20% . Before each assay, P. aeruginosa frozen stocks were freshly cultured to brain heart infusion agar and then incubated aerobically at 37 °C for 18–24 h.The ligands used are commercially available, i.e., TA , GA , DS  and AA . P. aeruginosa anti-biofilm, which was retrieved as a crystal structure (1K2Y) from the Protein Data Bank (PDB) (rscb.org/pdb) accessed on 23 August 2019. TLA was a PMM/PGM native ligand extracted from the 1K2Y crystal structure, which was used as a reference coordinate in determining the active site of PMM/PGM [https://pubchem.ncbi.nlm.nih.gov/ (accessed on 4 September 2019) that have similar structure to TLA, includes information about related compounds, chemical vendors, drug and medication information, food additives and ingredients, pharmacology and biochemistry, use and manufacturing, safety and hazards and toxicity. Ligands structure similar to TLA are hypothesized to have the same, or better, activity. The inhibitory activity of selected ligands against PMM/PGM were then screened and evaluated through molecular docking analysis and considered for commercial availability, so that they can proceed to in vitro experiment.The PMM/PGM S108A mutant was used as the target protein to design the  PMM/PGM . In addi+2 atoms from the molecular structure of PMM/PGM using Chimera version 1.13. The ligand geometry was optimized using Semi-empirical Quantum Method-Parametric Method 3 (SQM-PM3). Further optimization was performed with HyperChem 8.0 to calculate the electrostatic potential charge of each atom. Analysis was continually performed to determine the ligand and receptor parameters such as bonded, non-bonded, and charged using the AMBER FF14SB force field tool [PMM/PGM served as the receptor during docking. The receptor was prepared by extracting amino acid residues and Zneld tool  and Austeld tool  containevdW) and electrostatic energy (Eele).Molecular docking was performed in two stages, namely, redocking and docking of other test ligands, using the Dock6 package. During redocking, a cluster sphere with a radius of 10.0 Å was first selected to determine the native TLA ligand coordinates on the receptor active site. Next, a grid box with a grid spacing of 0.1 Å was created on the selected cluster sphere around the receptor active site. The native ligand RMSD value represented successful redocking (≤2.0 Å) . Next, bhttp://www.swissadme.ch/index.php) (accessed on 13 July 2020) and pkCSM (http://biosig.unimelb.edu.au/pkcsm/prediction) websites (accessed on 13 July 2020), with a 2D ligand structure (SMILES format) [The pharmacokinetic properties of each ligand were predicted using the SwissADME ( format) ,45 Drug-P. aeruginosa biofilm was assayed using the crystal violet method on a sterile flat bottom 96-well microtiter plate  and indicated as Minimal Biofilm Inhibitory Concentration (MBIC).The capability of the ligand to inhibit the extracellular matrix formation of P. aeruginosa inoculum 107 Colony Forming Unit (CFU/mL). The microtiter plate was incubated for 8 h to achieve irreversible adherence. Then, the medium was discarded and replaced with the ligands. Each well was filled with 100 mL of medium, and each ligand was investigated at twofold increasing concentrations. The starting concentrations were determined on the basis of the preliminary test result of each ligand, there were TLA (1.25 × 102–1.6 × 104 µg/mL), GA (3 × 101–2 × 103 µg/mL), DS (3 × 103–4 × 105 µg/mL), and AA (3.9 × 102–5 × 104 µg/mL).Each well was filled with 95 µL of Brain Heart Infusion (BHI) broth  and 5 µL of Next, the microtiter plate was incubated for 24 h at 37 °C. This assay used meropenem (Minimum Inhibitory Concentration/MIC: 0.5 µg/mL) as the positive control and bacterial inoculum as the negative control. The treatment was replicated three times.After incubation, the microtiter plate was washed with distilled water three times and then dried at room temperature for 5 min. Next, 125 µL of 1% crystal violet solution was added to each well and then incubated at room temperature for 15 min. The microtiter plate was washed three times under running water. Next, 200 µL of ethanol was added to each well ,46. OptiP. aeruginosa was cultured on 13 mm diameter nitrocellulose semipermeable membrane  and placed on an agar plate at 36 °C. The preparation of the ligand test was similar to the previous in vitro method. In total, 20 µL of bacterial suspension that reacted with the optimal concentration of each ligand was dripped onto a nitrocellulose semipermeable membrane layer on BHI agar and incubated for 24 h. The macroscopic morphology of the biofilm was observed on the membrane surface.P. aeruginosa biofilms were formed on a sterile 12 mm diameter round glass coverslip  in a 24-well polystyrene microtiter plate. The preparation of biofilm formation and ligand treatment was similar to the previous procedures in the in vitro biofilm matrix extracellular inhibition assay. The coverslips were submerged in 400 µL of bacteria and ligand suspension and incubated overnight at 37 °C. A biofilm grown without ligand treatment was used as the control. SEM preparation was performed in accordance with the method of Hess et al. and Kazmierczak [mierczak ,47 with P. aeruginosa. These potential anti-biofilm agents can reach almost 100% inhibition values at different concentrations. TLA, GA, DS, and AA had a biofilm inhibition rate of >50% at 2 × 103, 2.5 × 102, 4 × 105, and 1.25 × 104 µg/mL, respectively. Nitrocellulose membrane assay showed that controls and ligand-exposed biofilm had different appearances. SEM images revealed that the control biofilm could be covered in the EPS matrix. However, these images were not visible in treated biofilms. Molecular docking analysis showed that each ligand had an excellent binding affinity to the receptor, as supported by several hydrogen bonds . Furthermore, each inhibitor can be a promising drug candidate based on its pharmacokinetic properties. Analysis of drug-likeness and bioavailability showed good results, where all anti-biofilm candidates showed non-toxicity and met the following criteria: non-AMES toxic, non-hepatotoxicity, and non-skin sensitization.The in vitro experiments in this study proved that TLA, GA, DS, and AA had excellent activity against the biofilm of


Metabolism reprogramming plays an important role in tumorigenesis and osteosarcoma metastasis. Sine oculis homeobox 4 (SIX4) is reported to be a key transcription factor that is involved in glycolysis reprogramming of cancer cells. However, the role of SIX4 in osteosarcoma progression remains unknown. The expression profile of SIX4 in OS was evaluated in surgery samples of osteosarcoma patients. Functional studies were performed in vitro and in vivo. We found that SIX4 is significantly overexpressed in osteosarcoma and related to the undesirable prognosis of osteosarcoma patients. SIX4 promotes progression of osteosarcoma via upregulating isocitrate dehydrogenase 1 (IDH1), which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients. Our work provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.22.1A total of 24 cases of osteosarcoma tissues and the adjacent non‐tumour tissues were obtained from patients who underwent surgical excision at the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an No.3 Hospital, the Affiliated Hospital of Northwest University, Shaanxi, China. All patients were obtained informed consent, and this study was approved by the ethics committee of the Second Affiliated Hospital of Xi'an Jiaotong University.2.22. U2OS cells were transfected with pBabe‐SIX4 to generate SIX4 overexpression cells, while MG63‐shSIX4 cells were established by lentivirus infections with pLKO.1‐shSIX4.The human osteosarcoma cell lines U2OS, 143B, MNNGHOS, MG63, G292 and the normal osteoblast cell line hFOB1.19 were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 ng/ml) at 37°C with 5% CO2.3Osteosarcoma tissues and cell lines were collected and lysed in RIPA buffer with protease inhibitor  for 30 mins, followed by centrifugation for 15 min at 12,000 rpm at 4°C. The supernatants were collected, and the protein concentration was quantified by using BCA assay kit (Beyotime Biotechnology). A total of 30 μg of protein were separated by SDS‐PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked before being incubated with primary antibodies: anti‐SIX4 antibody ; anti‐IDH1 antibody ; anti‐β‐actin antibody . The corresponding horseradish peroxidase‐conjugated secondary antibodies were used, and the proteins were detected using chemiluminescent detection reagents and films.2.44 cells/200 μl in Transwell® cell culture insert  and allowed to migrate for 24 h. Then, the chamber was fixed with methanol and stained with crystal violet (0.1%) for 15 min. The cells on the upper surface of the filters were removed, and the cells that migrated through the filters were counted.Transfected osteosarcoma cells were cultured at 1 × 102.5Transfected osteosarcoma cells were seeded in a 12‐well plate with 80% of confluency. Vertical and horizontal lines were drawn on the bottom of each well by a sterile pipette tip and cultured for 24 hours. The same view of the scratch was captured, and the migration rate was evaluated by Image J software.2.6Ct−ΔΔ method was employed to analyse the gene expression.Osteosarcoma tissues and cell lines were collected, and the total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Real‐time PCR was done with SYBR Green Master mix (Takara). The 22.7Luciferase activity was detected using the Dual Luciferase Assay (Promega) according to the manufacturer's instructions. The transfected cells were lysed and centrifuged at maximum speed for 1 min. Relative luciferase activity was determined using a ModulusTM TD20/20 Luminometer (Turner Biosystems), and the transfection efficiencies were normalized according to the Renilla activity.2.8t‐test was used to compare individual data between two groups. One‐way anova test was used to compare the data among three or more groups. Pearson's correlation coefficient was calculated to show the relationship between the expression levels of SIX4 and IDH1 mRNA. The Kaplan–Meier method and the log‐rank test were used to compare patient survival. Data are presented as mean ± SD or median with interquartile range. p < 0.05 was considered statistical significance.Student's 33.1The upregulation of SIX4 mRNA and protein was further validated in fresh samples from surgery patients of osteosarcoma Figure . More im3.2To investigate the function of SIX4 in osteosarcoma, human osteosarcoma cell line MG63 and U2OS were used for SIX4 knockdown and overexpression, respectively Figure. . SIX4 do3.3Glycolysis is a key feature for tumorigenesis and metastasis, and SIX4 has been reported to be crucial for glycolysis.3.4To explore whether SIX4‐mediated osteosarcoma metastasis is associated with upregulation of IDH1, we examined the expression of IDH1 in SIX4 overexpression and knockdown cells. Overexpression of SIX4 leads to increased IDH1 mRNA and protein expression, whereas downregulation of SIX4 decreased IDH1 expression compared with the corresponding control cells Figure , suggest4Accumulating evidence has shown that glycolysis is a key feature for tumorigenesis and metastasis of osteosarcoma.Given the significance of SIX4 in carcinogenesis and metabolic reprogramming, we test the expression of SIX4 in osteosarcoma tissues and cell lines. Our results showed that SIX4 was upregulated in osteosarcoma, and its upregulation predicted poor outcomes for osteosarcoma. These findings suggested that SIX4 plays an important role in osteosarcoma. However, no evidence is available to demonstrate the functional role of SIX4 in osteosarcoma. Therefore, we generated SIX4 overexpressing and knockdown cell lines to further explore the role and mechanisms of SIX4 in the pathogenesis of osteosarcoma. We found that SIX4 is required for the migration of osteosarcoma cells. Thereafter, we evaluated the ECAR/OCR and glucose consumption to figure out whether the pro‐migration capacity of SIX4 is associated with glycolysis. As expected, both ECAR/OCR and glucose consumption were upregulated when SIX4 is overexpressed, while downregulation of SIX4 spontaneously decreased the ECAR/OCR and glucose consumption of tumour cells. All these findings demonstrated that SIX4 promoted osteosarcoma progression via regulating tumour cell glycolysis. Then, we analysed serious glycolysis‐related genes and found that IDH1 can be transcriptionally regulated by SIX4 to promote cancer cell migration. IDH1 is a key enzyme of TCA cycle that catalyses the conversion of isocitrate to alpha‐ketoglutarate.In conclusion, our study found that SIX4 promotes progression of osteosarcoma via upregulating IDH1 and metabolic reprogramming, which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.Bing Li: Investigation (lead). Xiaoqian Dang: Investigation (supporting); validation (supporting). Jiafeng Duan: Investigation (supporting); validation (supporting). Guangyang Zhang: Investigation (supporting); validation (supporting). Jia Zhang: Investigation (supporting); validation (supporting). Qichun Song: Conceptualization (lead); data curation (lead); supervision (lead).The authors declare no competing interests.Appendix S1Click here for additional data file.


Anethumgraveolens L.) is the most valuable medicinal seed spice crop of Apiaceae. It bears small yellow flowers in the form of umbels. Being a cross-pollinated crop, floral visitors play vital role in pollination and seed sets. Hence, the present study was conducted at the ICAR-National Research Centre on Seed Spices, Ajmer (Rajasthan), India to discover the pollinator’s community, foraging behaviour and abundance of most frequent pollinators and different modes of pollination on seed yield and quality of this seed spice crop. The insect visitors community of dill seeds was composed of 28 insect species belonging to 14 families of 6 orders. Most of floral visitors started their foraging activity at 8.00 h, reached peak activity between 12.00 and 14.00 h and their activity ceased at 18.00 h. Apisflorea,A.dorsata,A.mellifera, solitary bee, Halictus sp. and two unidentified species of Hymenoptera; Episyrphusbalteatus (DeGeer), Episyrphus sp., Eristalis sp and two other Musca species of Diptera were identified as potential and regular floral visitors of dill seeds. The highest seed yield of 1505.63 kg/ha was recorded in the treated plots provided with only 10% jaggery solution and was at par with the open pollination. A lower seed yield of 1432.5 kg/ha was recorded in plots pollinated only with A.mellifera inside insect cages. Open pollination with 10% jaggery solution spray increased the seed yield of dill seed crop by 57%, one-thousand seed test weight by 96% and the essential oil content by 27% over control plots. These results show that managed pollination is a much better way to enhance yields and quality of dill seed crop than other treatments including only honeybee-based pollination.Dill seeds ( Ollerton et al.3 reported that 87.5% flowering plant species are pollinated by animals, whereas, remaining plant species are either wind-pollinated or completely reliant on autonomous seed production5. Insect pollinators, particularly bee species visit flowers of angiosperm plants to collect nectar and pollens, thereby the visiting plants get pollinated and also enhance the production as well as quality. Non-availability of effective pollinators during flower anthesis leads to massive loss in the agricultural production. Gallai et al.6 reported annual value of pollination service to €153 billion in 2005, contributing 9.5% of agricultural food production crops. Further, the annual market value of additional crop production directly linked with pollination services is estimated at $235bn-$577bn worldwide7 In the absence of animal pollination, a potential annual net loss of economic welfare of $160 billion-$191 billion incurred globally2. In the last few years, there has been substantial increase in the value of this service due to decreasing numbers of insect pollinators, mainly honeybees because of lack awareness of their role in pollination among farming community and adverse impact of indiscriminate use of more toxic pesticides and changing climate scenario in the country. The reasons of pollinators decline are excessive use of pesticides10 disposable plastic cups12, destruction of natural habitation13 and intensive farming practices, mono-cropping and higher temperatures associated with climate change14.Pollination is one of the most important ecosystem services to agriculture, once 75% of the food crops and nearly 90% of wild flowering plants rely on animal pollination, in different degreesAnethumgraveolens L.) is an important aromatic as well as medicinal annual herb from the Apiaceae family. Seeds and leaves of this plant are used as the main edible parts15 and it is a widely used spice due to its pleasant spicy aroma and plenty of nutritional and medicinal properties18. Dill essential oil contains dill apiole, carvone, carvacrol, dihydrocarvone, limonene, p-cymenand α-phellandrene15. It is used in the treatment of several ailments viz., gripe water to relieve colic pain in babies and flatulence in young children19, while the seed is carminative, mildly diuretic, galactogogue stimulant and stomachic20. It is also used in the cure of certain urinary complaints, piles and mental disorders21. In India during 2019–2020, dill was cultivated in an area of 32.79 thousand ha with a production of 34.56 thousand tonnes seed and a productivity of 1054 kg/ha22. Anethumgraveolens is a cross-pollinated crop, with small yellow flowers, diploid (2n = 22) and strongly protandrous. The inflorescence is a compound umbel, 4–16 cm in diameter and flowers bloom in a strict sequence. The main umbel is the first to bloom followed by different range umbels in order of their range23. Flowering is normally completed in 9–12 days and anthers dehisce in the morning and remain receptive until midday depending upon ambient temperature24. Flowers are homogamous and hermaphrodite. In the umbel, some flowers are entirely pistillate and some staminate, and a few are hermaphrodite. Primary umbels bear hermaphrodite flowers, whereas, the secondary and tertiary umbels bear hermaphrodite ones on the margins and staminate types in the centre25. The ovary of the pistillate flower contains two ovals. Yellow staminate flowers have five stamens which arise between the petals. The staminate flowers contain noticeable quantity of nectar and have a strong odour25, attracting mainly bees, flies, and other pollinators. Honeybees, particularly Apisflorea have been documented as the most dominant floral visitors of dill flowers26.Dill  of 2016–2017 and 2017–2018. The study site is surrounded by Aravalli hills, located at the coordinates 26°27′0″ N latitude, 74°38′0″ E longitude, with 460 m msl altitude2 (5 × 4 m) under 50 × 30 cm crop geometry (row to row and plant to plant spacing) following recommended package of practices for dill seeds39. The five treatments as independent variables namely T1-without insect pollination (WIP-caged) as control, T2-open pollination (OP), T3-bee pollination (BP-caged), T4-open pollination with jaggery solution 10% and T5-open pollination with sugar solution 10% were applied as different modes of pollination. Jaggery and sugar solution were sprayed on the flower canopy of dill seeds at 20–30% flowering initiation stage to attract bees and other floral visitors for pollination. Plants of two plots (WIP and BP) in each replication were covered by insect cages made of GI pipes, lined with 16-mesh insect proof nylon nets. The insect cages measuring of 5 × 4 × 3.5 m (length × width × height) were installed on plants before initiation of flowering40 to kept plants free from the activities of any insect species within the insect cage of control plots (WIP) in each replication throughout the flowering period. At the same time, a four-framed colony of A.mellifera was kept inside the cages of bee pollination plots (BP) in all replications to ensure pollination services, while the plants in open pollination plots were exposed to natural pollination by all floral visitors. It is confirmed that all methods were performed in accordance with the relevant guidelines/regulations/legislation.The field experiment was designed to study the diversity of different floral visitors, dynamics of foraging activity, abundance of potential pollinators and effect of different mode of pollination on yield and quality of dill seeds in semi-arid region of the country. The field trials were laid out in a randomized block design with five treatments, replicated four times Fig. . Dill se44. Observations on floral visitor diversity consisted sweep-netting the floral visitors throughout the blooming period40 i.e. from the first week of January to the last week of March. The sweep net catches were recorded each time in open pollination plots in all the four replicates, where no plant protection measures were taken throughout the cropping period. In each observation, 4 to 5 sweep-net catches were made for 10 min on 20 square meters of flowering area of the entire plot/replication. These observations were made five times at 09.00, 10.00, 12.00, 14.00 and 16.00 h at weekly intervals throughout the flowering period. Floral visitors were also recorded at same time interval by visual counting from 1 m2 flower canopy to determine the mean abundance/m2 per day and proportion (%) of individuals from total floral visitors. The observation timing was delayed till 09.00 h during the period due to heavy fog deposited on plant canopy and there was lack of bright sunshine. Specimens of floral visitors were collected, preserved and identified based on available literature information, but all specimens are not assigned to species level. The identifications of the insects were confirmed by the taxonomist.For this study, data on floral visitors’ diversity and their population effectiveness were recorded by sampling methods of direct observation and sweep net collection2 bloom area per five minutes) at weekly interval throughout the flowering period. Observations of pollinator’s flower visitation were conducted by scan sampling methods45. The data on number of floral visitors/m2 bloom area per five minutes was recorded four times  per day at weekly interval and then converted to a mean population. Similarly, the temporal abundance of 10 most important and frequent floral visitors was studied on dill flowers. Abundance of flower visitors was studied as the number of floral visitors/m2 boom canopy per minute to determine their foraging rates during particular period of the day. Observations on number of floral visitors/ m2 boom canopy per minute were recorded at hourly intervals from 06.00 to 10.00 h and two hourly intervals from 10.00 to 18.00 h for 10 calm and clear sunny days43 during peak flowering stage.The insect species visited flowers and transport pollen grains in the body were considered as pollinator of dill seeds. Population dynamics study of selected floral visitors was conducted to know the foraging activity and temporal abundance on dill seeds flowers. Foraging activity of 14 different floral visitors was recorded as population fluctuation  was also studied as the number of flower umbels visited per minute46 as well as the number of plants visited per five minutes, where the observations were recorded six times a day  for 10 calm and clear sunny days to determine their potentiality within species as pollinator of dill seeds for semi-arid region of the country.The foraging rate of 3 honeybee species  per replicate were sprayed with jaggery solution 10% and sugar solution 10% as indigenous bee attractants at 20% flowering stage49 to increase Apis pollinators activity up to 3rd day of treatment. Data on yield parameters were recorded on randomly selected five plants /plot and on the total number plants harvested separately for each mode of pollination. For the assessment of quality of dill seeds in terms of test weight and essential oil contents, 1000 clean seeds were randomly selected using seed counter machine and test weight was measured using physical balance. The essential oil was extracted from 30 g dried dill seed using hydro-distillation method50.Pollination effectiveness was measured in term of per unit area yield appreciation, one thousand seed test weight and essential oil contents. The five different treatments (modes of pollination) as described in the sub-heading experimental design were followed in all four replications to determine the impact on seed yield2 bloom area per five minutes), and temporal abundance  were recorded separately for two years and then converted into pooled analysis to represent the population dynamics. Similarly, seed yield per plot were recorded separately for two consecutive years and converted into kg per hectare yield, test weight per 1000 seeds and essential oil content in percentage. After conversion into appropriate unit, data of both the years for each parameter were pooled and mean values for yield and seed quality  were subjected to statistical analysis using analysis of variance (ANOVA). Means were compared by use of Duncan’s Multiple Range test (DMRT) at P ≤ 0.05.The statistical analysis was performed using the SPSS statistical software. We calculated mean population of each floral visitor per day for entire flowering period and their per cent proportion of total floral visitors to study the diversity of insect pollinators of dill seeds. Weekly population fluctuation and abundance of potential and frequent floral visitors on dill flowers were also studied by calculating means, SE (m) and p value by ANOVA analysis using statistical software OPSTAT developed in Haryana Agricultural University, Hisar, India. The data on population fluctuation  followed by Diptera with eight species, while three species each were from Hemiptera, Coleoptera and Lepidoptera and one species was from Neuroptera order  followed by A.mellifera (32.6 bees), Apisdorsata (19.6 bees), and Halictus sp. (9.81 bees), on dill flower ecosystem. The other Hymenoptera species recorded were Polisteshebraeus,Ceratinasexmaculata Smith,Xylocopa sp., Camponotus sp. and unidentified hymenoptera sp 1, but their numbers were negligible. A.florea and A.mellifera were the most frequent and dominant floral visitors constituting 43.69 and 22.76% proportion of total Hymenoptera and 30.14 and 15.71% proportion of total floral visitors, respectively. The second most dominant group of dill floral visitors was eight species from Syrphidae and Muscidae families of Diptera. Out of these, Episyrphusbalteatus (DeGeer) and Episyrphus sp. (Syrphidae) and Muscadomestica (Muscidae) were only recorded as the part of the pollinator’s community and the most abundant with mean population of 12.96, 6.33, and 6.09 flies /m2 per day, respectively  and Raphidopalpafoveicollis Lucas (Coleoptera), Bagradahilaris Burm. (Hemiptera), Chrysoperlazastrowisillemi (Esben-Petersen) (Neuroptera), Helicoverpaarmigera Hub., Pierisbrassicae L. and an unidentified species (Lepidoptera), however their numbers were negligible (0.3 to 1.1 insects/m2 per day).The floral visitors of A.florea,A.mellifera,A.dorsata and Halictus sp. were the most dominant and regular pollinators in the region. The highest activities of different bee pollinators were recorded in the range of 10 to 122 bees/m2 bloom area/5 min with a mean of 45.2 ± 45.5 (± SD) in the third week of February. Honeybee species A.florea (122 bees/m2 bloom area/five minutes) were noticed as most frequent and statistically highest populated pollinator on dill seeds which was followed by A.mellifera,A.dorsata, Halictus sp. and C.sexmaculata , respectively in the third week of February. Among the Syrphid pollinator, species Episyrphusbalteatus,Episyrphus sp., Eristalis sp. 1 and Eristalis sp. 2 were recorded in the range of 6.2 to 20.2 flies/m2 bloom area/five minutes with different peaks and comparatively less populated regular floral visitors  and frequency. The visiting frequency increased gradually with the higher temperature and more sun light reaching peak population between 12.00 and 14.00 h showing the population in the range of 2.10 to 20.63 insects/m2 bloom area per minute at 14.00 h. Apisflorea was the most abundant (20.63 bees/m2 bloom area/minute) pollinator followed by A.mellifera (15.45 bees/m2 bloom area/minute) and both the pollinators were statistically differed in abundance (P ≤ 0.05) with a mean value of 7.61. Among the bee species, only A.dorsata sustained its activity throughout the day, starting at 07.00 h and reaching the peak (8.05 bees/m2 bloom area/minute) at 12.00 h followed by a decline to negligible number (0.4 bees/m2 bloom area/minute) at 18.00 h. The abundance of A.dorsata on dill seeds was statistically lowered (P ≤ 0.05) to A.mellifera and A.florea but higher to remaining floral visitors at all intervals. Halictus sp. started their activity at 08.00 h, then steadily increased and reached to its highest population (3.6 bees/m2 bloom area/minute) followed by a completely checked out after 16.00 h. Syrphid species were available in the field of dill crop throughout the day, either sitting or flying on flower umbels and exhibited a population fluctuation pattern. Episyrphusbalteatus, Episyrphus sp. 1 and Eristalis sp. started their activity at 07.00 h with very few numbers (0.05 to 1.00 flies/m2 bloom area/minute), thereafter their population fluctuations were statistically varied (P ≤ 0.05) depending on species with their peaks at different hours of the day  followed by A.mellifera and A.florea which visited 5.43 and 3.88 flower umbels per minute, respectively at 14.00 h. Thereafter, there was a reduction in flower visiting frequency of honeybee pollinators  followed by A.mellifera (3.58) and A.florea (2.60) during 14.00 h  honeybee species under cage condition . Among the four modes of pollination, significantly lowest seed yield of 1317.5 kg/ha was recorded in sugar solution 10% treated plots. Overall, the four modes of pollination had a significant impact on seed yield showing 37.4 to 57.04 percent increase over control. Similarly, the quality traits  of dill seeds were also influenced by different modes of pollination. All pollination treatments were statistically at par (3.16 to 3.34 g) for test weight within treatment but significantly better to control . Insect pollination also increased the essential oil having carvone and dill apiole content from 1.71% in control to 2.16–2.19% in the treatments but statistically at par with each other , respectively . In the study area, Apisflorea was recognized as the most frequent and dominant pollinator of dill followed by A.mellifera and A.dorsata. Earlier, Chaudhary and Singh47 reported that A.florea was the prominent pollinating agent in different seed spice crops under arid to semi-arid regions corroborates to the present study. The honeybee species Apismellifera,A.dorsata and A.florea were also reported as major and potential pollinators of coriander53, fennel54, cumin38 and black cumin56. Abundance and composition of pollinators vary with the varied geographical area, latitude and the time57. In our study, the other pollinators recorded to visit dill flowers were true flies Episyrphusbalteatus, Episyrphus sp., and Eristalis sp. 1 and Eristalis sp. 2 from Syrphidae and four species from Muscidae. Previous studies have reported syrphid flies to be good pollinators of many crops such as coriander47, fennel58 and many other seed spice crops59. In this study, more than two-third of the total floral visitors in dill crop ecosystem belonged to Hymenoptera and Diptera and hence referred to as majority pollinating insects for seed spices.Typically small, actinomorphic yellow flowers of dill seeds produce plant resources like nectar and pollen to attract wide range of insects, bees and waspThe flowers of dill seeds received floral visitors in first week of January with lower numbers in our study due to flowering initiated on crop at this stage. The higher populations of the majority of floral visitors were noticed between second and third weeks of February due to peak of flowering and increased availability of nectar and pollen. The peak activities of these pollinators varied among species due to their foraging nature and availability of nectar and pollen in the flowers. No literature available on this aspect particularly on dill seeds, hence, it could not be discussed in length.53 observed that peak activity during this period was also due to visual impact of the compound umbel along with presence of exposed nectar and availability of pollen. In diurnal dynamic pattern, the hymenopterans, A.florea, A.mellifera and Halictus sp. were more abundant at 14.00 h, as most of the bees consume nectar and its availability increased after 12.00 h. However, wild honeybee A.dorsata visited dill flowers throughout the day with higher numbers at 12.00 h. Kapil et al.60 and Bhalla et al.61 also reported similar findings on different crops including seed spices. Foraging activities of other floral visitors in the present study exhibited an indefinite pattern. Episyrphusbalteatus, Episyrphus sp and Eristalis sp 2 foraged in sufficient numbers for longest period till 17.00 h, while none of floral visitors except Musca sp. 2 was observed foraging at 06.00 h on A.graveolens flowers in winters under semi-arid region of Rajasthan, India. Continuous foraging activity of Musca sp. was reported on coriander from morning till evening (06.00 h–18.00 h) during winter47, whereas Sikdar et al.59 noticed the higher activity of dipteran flower visitors during the morning (09.00 h–10.00 h).The activities of most of the floral visitors on dill seeds flowers were initiated at 8.00 h and peak activity of most of the pollinators were observed from 12.00 to 14.00 h in our study, may be due to the availability of nectar and pollen was highest during this period. Koul62, sugar concentration in nectar63, length of proboscis and instinctive foraging dynamics64 and some abiotic factors i.e., wind velocity, ambient temperature, relative humidity and intensity of light65. In this study, the highest visitation frequency on dill flowers was recorded for A.dorsata while A.florea had low visitation rate and greater stay time with highest pollen deposition because of small size and better foraging habit for seed spices. The higher foraging rate of pollinators enhanced the pollen rubbing of flowers and simultaneously increased the seed set and seed maturity rate in dill. Singh et al.66 reported that higher foraging rate and visitation frequency created higher chance of pollination, whereas Engel and Irwin67 observed that there was no standard rule and the insects with high visitation rates could be poor pollen depositors. In another study, A.florea was rated as lower forager but a more successful pollinator for different seed spices26.Foraging rate and flower visitation frequency of insect pollinators on cross pollinated crops are important to judge the pollination efficiency of different species. Earlier researchers also reported the dependency of floral visitors foraging rates and visitation frequency on various factors including quantity of floral rewardsA.graveolens) were influenced significantly by the managed pollination services through various floral visitors. Sihag68 found that insect pollinators significantly enhanced the yield and quality of seeds in cross pollinated cruciferous and umbelliferous plants. In the present study, 57 per cent higher yield of dill seed was obtained in open pollination with 10% jaggery solution spray treatment over control due to additional input of other pollinators allowed from natural sources during entire flowering season. Bee pollination with A.mellifera under insect cage condition also appreciated the seed yield of dill seeds by 49.4% over control plots shows importance of bees alone for pollination in this seed spice crop. Meena et al.38 found in their earlier study that bee pollination increased the yield of cumin by 40.03%. The number of seeds set per plant in dill was significantly greater in open plots than control (caged plots). Verma and Dwivedi69 also concluded that insects visiting the flower enhance the rate of fruit setting by promoting cross pollination. Pollination management in various cross pollinated crops through honeybees and true flies, resulted in higher fruit set, seed set, test weight and oil content71 and these reports support the findings of the present research. In our study, jaggery and sugar solution 10% used as indigenous bee attractants, where jaggery solution 10% increased the seed yield significantly as compared to other treatments but at par with open pollination. Spraying of 10% jaggery solution enhanced the activity of A.dorsata,A.florea and A.cerana on Alliumcepa48 and A.cerana on buckwheat49. The impact of managed pollination on improvement of the seed quality parameters in dill over control conforms to the findings of Sihag68 that pollinators greatly influence seed size, test weight (weight/1000 seeds) and seed germination percentage in cruciferous crops. Proper pollination can improve the quantity and quality of fruits, nuts, oils, and other crops produced72. Meena et al.38 also recorded higher essential oil content in cumin seeds of bee pollinated plants than control plots.The seed yield and quality of dill seeds (The investigation indicated that a total of twenty eight insect species visit the flowers of dill seeds crop throughout the flowering season in semi-arid regions of the country. Favourable time for foraging activities of most of the floral visitors is between 12.00 and 14.00 h. It is concluded from the present study that the honeybees for sure are good pollinators for the crop, and hence, there is need to conserve by enriching their habitat. We also recommend the need-based use of safer insecticides for the control of pest insect like aphid prior to flowering which is least disruptive to pollinators in the dill seed crop. If pollinator’s performance is poor, even during peak of flowering, a spray of 10% jaggery solution can be recommended to dill producers to attract pollinators. Studies on further quantification of their impacts and ways to enhance their utility in pollination management will help increasing dill production and productivity in semi-arid regions.


Salmonella typhimurium.The regulation of the synthesis of L-tryptophan (L-Trp) in enteric bacteria begins at the level of gene expression where the cellular concentration of L-Trp tightly controls expression of the five enzymes of the Trp operon responsible for the synthesis of L-Trp. Two of these enzymes, trpA and trpB, form an αββα bienzyme complex, designated as tryptophan synthase (TS). TS carries out the last two enzymatic processes comprising the synthesis of L-Trp. The TS α-subunits catalyze the cleavage of 3-indole D-glyceraldehyde 3′-phosphate to indole and D-glyceraldehyde 3-phosphate; the pyridoxal phosphate-requiring β-subunits catalyze a nine-step reaction sequence to replace the L-Ser hydroxyl by indole giving L-Trp and a water molecule. Within αβ dimeric units of the αββα bienzyme complex, the common intermediate indole is channeled from the α site to the β site via an interconnecting 25 Å-long tunnel. The TS system provides an unusual example of allosteric control wherein the structures of the nine different covalent intermediates along the β-reaction catalytic path and substrate binding to the α-site provide the allosteric triggers for switching the αββα system between the open (T) and closed (R) allosteric states. This triggering provides a linkage that couples the allosteric conformational coordinate to the covalent chemical reaction coordinates at the α- and β-sites. This coupling drives the α- and β-sites between T and R conformations to achieve regulation of substrate binding and/or product release, modulation of the α- and β-site catalytic activities, prevention of indole escape from the confines of the active sites and the interconnecting tunnel, and synchronization of the α- and β-site catalytic activities. Here we review recent advances in the understanding of the relationships between structure, function, and allosteric regulation of the complex found in  L-Trp synthesis is tightly regulated in enteric bacteria, and this control begins at the level of gene expression  (7) β-hydroxyl by indole yielding L-Trp and a water molecule  , while tmolecule . During molecule . This chmolecule . Within of L-Trp .Salmonella typhimurium bienzyme in complex with substrates, and with a variety of substrate analogues and/or covalent intermediates bound to the α- and β-sites  tes viz.  and allotes viz. . Recent tes viz.  and NMR viz. 19F  and 17O O probes . Solid sched PLP , and β-sresidues  and moleresidues  have bec2 group of βLys87 plays essential roles in the formation of the internal and external aldimines of substrates L-Ser and L-Trp , giving E(Ain) and releasing α-aminoacrylate. This highly nucleophilic three-carbon enamine then reacts to form a new C-C bond with the E(Ain) PLP C4′ carbon (R state (PDB ID: 2J9Y). The α-aminoacrylate side reaction has been reported for several other PLP-dependent enzymes that involve α-aminoacrylate Schiff base intermediates  bound to the α-site, the TS internal aldimine and the L-Ser external aldimine give complexes with closed α-subunits and open β-subunits  and the structures of the E(Aex1) and E(Aex2) species formed with the βLys87Thr mutant give αRβR complexes  is formed activates the α-site by ∼30-fold while the β-subunit is activated about 10-fold  is a relatively hydrophilic region extending from the indole ring subsite of the α-subunit into the β-subunit to tunnel residues βTyr279 and βPhe280  is a very hydrophobic, dewetted nanotube and extends from βY279/βF280 into the indole ring sub-site of the β-catalytic site. There are no waters detected in this region of the tunnel  in a bienzyme complex. . The inte280 T1,   site provides an essentially non-aqueous environment for the C-C bond forming step between the indole ring C3 and the α-aminoacrylate Cβ carbons. While it previously has been speculated that βPhe280 plays a gating role in the channeling of indole  is very fast relative to the turnover rate of the β-rection, as is the rate of reaction of IGP with E(A-A)  . Since th E(A-A) .via synchronization of the α- and β-reactions  bound to the TS metal coordination site activate the α2β2 bienzyme complex, including Na+, K+, Cs+, NH4+ and guanidinium ion (10)  . The ava+ or Cs+  T and E(Aex1)T and Cs+ favoring E(A-A)R and E(C)R  and two waters, whereas Cs+ nearly fills this cavity and typically coordinates to 5 or 6 of the carbonyl oxygens). The K+ complex incorporates three of the carbonyl oxygens and a single water R . Inspectnd E(C)R  which inle water b. Of spsub-site , 6A–D. C T-state  while Cs1) with E(A-A) is modulated by proton binding and involves two groups with apparent pKa values of ∼7.8 and ∼10.3. Since this pH dependence is not observed in the β2 dimer 2AP and E(A-A) have deprotonated Schiff base nitrogen states as the dominating tautomers (T conformation while the E(A-A)R and E(C3)R conformations are significantly more dehydrated  the PLP moiety has a protonated Schiff base nitrogen  Caulkin, whereasautomers  (Caulkinautomers . We posthydrated .The PDB archive  shows thTβT and ∼500-fold in αTβR). When the α subunit is switched to the closed conformation (R state), loop αL6 (residues α179–α193) folds down over the catalytic site and makes interlocking interactions with loop αL2 (residues α56–α60) and helix βH6, thus creating a local environment where the ligand is shielded from solution, and access from solution into the interconnecting tunnel is blocked, TβT, αRβT, αTβR and αRβR (14) and N-(indolinyl) 3-glycerolphosphate   , 8 proviNGP, 15) .2β2 TS complex found in Pyrococcus furiosus TS shows the α-subunits with the closed conformation and a well-ordered αL6 loop  that prevent the escape of indole from the enzyme α-site and the tunnel, creates the indole sub-site at the β-catalytic site, and blocks the transfer of L-Ser and L-Trp between the enzyme β-site and solution.In contrast to the α-subunit, the β-subunit allosteric transition involves a relatively modest conformational change wherein an 88 amino acid residue domain  moves as a rigid body, undergoing a slight rotation and translation. This motion causes a displacement of COMM domain residues by ∼ 2.6 Å along the αβ subunit interface and by ∼ 4.5 Å near the β-catalytic site . While tormation , 2003 , E(C1), E(A-A), E(C2), E(C3) and E(Aex2), synchronizes the catalytic cycles of the α- and β-reactions, and prevents the escape of indole from the confines of the α and β sites and the tunnel during the αβ catalytic cycle  and E(A-A) species is accompanied by a switch from the T state to the R state. Early work (1) and E(Aex2) intermediates strongly in favor of structures wherein both the α- and the β-subunit reside in closed conformations, i.e., αRβR. Thus, RβR state. Studies by R(A-A), thus shifting the distribution of β reaction intermediates in favor of ET(Aex1) and an altered substrate specificity. Both the βAsp305Ala and βArg141Ala mutants give distributions of intermediates that are shifted in the favor of ET(Aex1). Recent studies (R(C) species, while, with the βGln114A mutant, these reactions are significantly impaired and ASL binding shifts the population from the ER(C) species to a mixture of the ET(Aex) and ET(GD) complexes  (18) (19) (2) and E(C3) in the β-reaction. The x-ray structures also provide additional information about the allosteric states that predominate along the β-reaction pathway. The complexes of OIA and DOA give mixtures of both the external aldimine and the carbanion species within the same crystal while the subunit conformation is αRβR   and anal17) (18)  and DOA 18) (19)  recently18) (19)  (Phillip18) (19) . These s is αRβR . L-Trp g is αRβR  with theormation  whereas mediates .in vivo, the TS β-site exists within the bacterial cell primarily as the quasi-stable α-aminoacrylate species in the αTβR conformation. In the absence of IGP, the α-site has the open αT conformation, thus giving a TS resting state, αTβR, where the β-site is activated but sequestered away from small molecule nucleophiles that could cause deleterious side reactions  via E(C1) to E(A-A) at the β-site triggers activation of the α-site ∼30-fold (R conformation until βR is switched back to βT when E(C3) is converted to E(Aex2)  . Notice T and βR is to cause an in-phase synchronization of the α- and β-reactions  and the allosteric units predominately αTβR  giving E(C2) which is quickly converted to E(C3) while the allosteric unit remains in the completely closed state, αRβR (2), the allosteric unit switches to the αTβT state releasing G3P. Synthesis of L-Trp is completed when E(Aex2), via E(GD2), is converted to the E(Ain)(L-Trp) complex followed by the release of L-Trp from the βT conformation of E(Ain)  is formed via the reaction of E(Ain) with L-Ser to give E(GD1) and E(Aex1). Since E(C1) is only detected as a fleeting intermediate (1) is unknown. It is clear that the quasi-stable E(A-A) has the βR conformation and the α-site is activated. Just as the allosteric switching in hemoglobin is essential to the transport of dioxygen, CO2 and H+, the allosteric switching in tryptophan synthase is essential for the efficient synthesis of L-tryptophan from IGP and L-serine.Carbon-carbon bond formation at the β-site then occurs te, αRβR , 2, 14. f E(Ain) . At thisrmediate , the con1) is converted to E(A-A) via E(C1), βT is switched to βR, the α-site is activated ∼30-fold and the indole sub-site of the β-site is expanded to dimensions tailored to match the VDW dimensions of Indole  rotates to a position where βLys87 functions as the acid catalyst to facilitate the elimination of the E(C)1 hydroxyl to give E(A-A)  is converted to E(C1). It is not clear if these conformational changes in the site residues and the reacting substrate occur in βT or βR. Answering this question will bring into sharper focus an understanding of the linkages between the α- and β-subunit allosteric transitions and the chemical transformations at the α- and β-sites. A second, related issue arises concerning the formation of the indole sub-site in the β-subunit. The indole sub-site of the β-subunit is too small in the βT complexes of E(Ain) and E(Aex1) and only expands to dimensions matching those of Indole when βT is switched to βR. The mechanism for this allosteric switch remains an elusive and open question, and requires further attention to determine how the allosteric switch to βR triggers the expansion of the indole sub-site to fit the dimensions of indole, and why the sub-site dimensions are linked to the allosteric transition.With the wealth of structural and functional data now available, the larger picture of allosteric control in tryptophan synthase is coming into focus. Yet questions remain, particularly on the coupling of structure to the finest level chemical details of the mechanistic transformations. At the chemical level, the linkage between α-site activation and E(A-A) formation is only partially understood. When E  (Holmes e E(A-A) . In thisin situ rather than depend on the surrounding milieu as a source of L-Trp. In some higher organisms , indole is utilized both for the synthesis of L-Trp and as a starting point for the synthesis of a plant secondary metabolite  for the synthesis of L-Trp in enteric bacteria. The integrity of the synthetic pathway is ensured by the protection of both indole and E(A-A) from deleterious side reactions by confining indole to the environs of the α-, and β-subunits and the interconnecting tunnel, sequestering the E(A-A) intermediate from reactive solvent and solute species, and synchronizing the α- and β-reactions to achieve efficiency. This portrait of L-Trp synthesis implies the existence of a strong evolutionary imperative in enteric bacteria to synthesize L-Trp tabolite . In thestabolite .Consequently, the TS allosteric transitions provide an interesting combination of the switching of a loop between disordered and ordered states in one subunit coupled to a small motion of an 88 amino acid domain in the other subunit of the heterodimeric allosteric unit. This regulation of the final two enzymes in the biosynthesis of L-Trp provides an elegant example of the relation between structure and function in a channeling nanomachine.


For KKS investigation in human plasma, this cascade is usually triggered on ice eventually by mixing with purified proteins. It has been established that purified FXIIa, PK, and nHK required a fixed order and timing for mixing protein on ice to ensure reproducibility of testing, we investigated the activation kinetics of both enzymes. The activation process of this kcat = 0.133 ± 0.034 and 0.0119 ± 0.0027 s−1, KM = 672 ± 150 and 115 ± 24 nM, respectively. The progress curve analysis of our in vitro KKS reconstitutions differed from a Michaelis–Menten mathematical simulation by a faster initial rate and a slower late rate. These two features were also observed ex vivo by using dextran sulfate-activated plasma and could reinforce the hypothesis of a maximal local effect (bradykinin release) and a minimal systemic consequence (PK preservation) in KKS activation process.FXIIa and PKa were found five-times less active on ice than at 37°C: ex vivo investigation of KKS in samples from patients presenting with hereditary angioedema and other inflammatory conditions.Analyzing the complete curve of cold KKS activation would provide valuable information for  KM) and the turnover number (kcat) remains partly established (t = 0) = [PK]0, [nHK](t = 0) = [nHK]0, [cHK](t = 0) = [cHK]0, [PKa](t = 0) = 0, and [F12a](t = 0) = [F12a]0, where [X] denotes the concentration of ‘X.’ The studied reaction displayed a faster start and a slower end rate than the MM model. The end of reaction rate was addressed by considering a competitive (Reaction 1) and an uncompetitive (Reaction 2) inhibition by cHK replacing (R1a) and (R2a) by: In the MM model, the two steps in  were iteinib, KI1, and KI2 are unknown parameters.The reaction start rate was addressed by supplementing the initial conditions above with a presteady-state initial burst, inib, KI1, KI2 …), which minimized SSD; iterations were stopped when the two first digits of each parameter did not change . Equations were programmed using R software, ‘simecol’ and ‘ggplot2’ packages ,19: a 30kcat and KM of PKa using nHK as substrate at 0°C, a temperature used in earlier KKS investigations [kcat/KM showed values only five-times lower than at 37°C, indicating that PKa was active from 0°C to 37°C . After limited contact between FXIIa and PK on ice, the reaction was slowed down by a 20-fold dilution and stopped by an addition of CTI as FXIIa inhibitor. We found that 5 mg/ml CTI nearly fully inhibited FXIIa, while PKa standard activity was decreased by less than 10% (not shown). Linearization of curves of PKa formation kinetics did not cross the origin of coordinates . The initial starting concentration set, called ‘1/6/6,’ was therefore ≈10−7 M PK and nHK, with a 1:1 ratio and a FXIIa concentration six-times lower, as assayed in prior experiments [A slow, but measurable, activity of both FXIIa and PKa enzymes on ice prompted us to follow the progression of the minimal proteolytic cascade made up of FXIIa, PK, and nHK and used in prior experiments . The streriments . ProgresUsing the R software and ‘simecol’ library ,19, a simax of the Michaelian steady state, linked to a fast-initial turnover of the enzyme. Extension of the linear section of product formation versus time curve crossed the origin of x-axis at an ordinate that is proportional to enzyme concentration [To address the first issue , we considered the possibility of an initial burst: in this presteady-state condition, substrate consumption is faster than Vntration ,22. inibWe evaluated the quality of these models by SSD between experimental points and theoretical curves, and found that SSD values were nearly halved, between that observed from MM and dIB models, i.e. from 17.95 to 8.53 . The initial burst coefficient of the first reaction was 0.39, as if 40% of FXIIa content rapidly activated one PK equivalent at the start of the reaction. The optimized value of this parameter was rather constant across all different models (SuppMat5).KD ≈12 nM and in a ≈1:1 stoichiometry [kCt in M7 (SuppMat1). Second, we considered the three steps of the serine protease reaction, with both intermediate and acyl-enzyme-forming steps faster than the last one, a necessary condition for initial burst [ke1 and ke2 in M21, 24 (SuppMat1). These models gave unlikely values of the intermediate kinetic constants without improving the SSD-fitting criteria (SuppMat5).In contrast, the second initial burst coefficient, calculated for nHK cleavage by PKa, was 1.64, considered as an implausible value, so we looked for more mechanistic models. First, since nHK and PK are complexed together in human plasma with a hiometry , we hypoal burst , i.e. kein vitro reaction on ice has been stopped by pipetting up and down sample in loading buffer at room temperature. A delay in stopping the reaction would account both for the initial faster rate in the progress curve analysis of both reactions and the intercept value in inib1 = 0.30, inib2 = 1.06, Δt = 38 s). These values were realistic and could be, respectively, interpreted as (i) an initial burst in reaction 1; (ii) an immediate cleavage of one nHK molecule by one newly formed PKa equivalent, either by initial burst or by preferential cleavage within some pre-existing PKa-nHK association [inib2 at its maximal value of 1, with a subsequent optimized time offset of 44 s . Thus, we suggest that a stopping delay and unidentified mechanisms of the initial burst accounted for the faster initial reaction velocity in the progress curve analysis.The ociation ; (iii) aWe next considered the apparently slower reactions than predicted after the first minutes. Since there was no carrier protein in reaction buffer, a FXIIa protein amount could hypothetically bind onto plastic despite the coating procedure and thus became inactive. We did not detect diluted FXIIa inactivation on coated tubes, or after 30 min on ice. A 0-rate degradation reaction of FXIIa, combined with initial burst, would end with 25% of FXIIa activity only, after 30 min, to marginally improve the SSD . This ruled out a decay of FXIIa as the explanation of the slower end of reaction.inib and KI1 , so we excluded the performance of the inhibition model to be artefactually linked to the selections of tested initial concentrations.An inhibitory function of cHK, the end product of reaction, on both FXIIa and PKa enzymatic activities has been next hypothesized, which effectively fitted models to experimental data, lowering the SSD value from 8.43  to 6.37 for competitive inhibition of FXIIa and uncompetitive inhibition of PKa . To ruleIt has not been possible to conclude on the inhibition mechanism : the simulation gave similar SSD and KI-fitted values within the ranges of 11–200 and 175–400 nM against FXIIa and PKa . In order to decipher the interaction between cHK and both enzyme activities, we added cHK in the current experimental set up used for FXIIa reaction kinetics: cHK also alleviated CTI inhibition of FXIIa, preventing an effective end of PKa activation and data analysis. We also added four-times more cHK than nHK prior to measure PKa kinetics, but this blurred the cleavage evaluation of the initial nHK, beyond the low precision of a western blot quantification. We concluded that investigation of inhibition of KKS by cHK requires another experimental design.in vitro reconstitution. In condition of plasma activation by DS [We wanted to formally compare KKS activation between human plasma and the on by DS ,16, FXIIon by DS . Human pAs shown in ex vivo and functional FXIIa in vitro. A delay of ex vivo activation of PK in plasma  occurred within a few minutes in ex vivo from FXIIa control by C1-INH, by other serpins or by α2-macroglobulin [in vitro at low FXIIa concentration  were also found in ex vivo DS activation in human plasma samples . Likewise, with measured Data from human plasmatic enzyme activities at 0°C are scarce. Outside of KKS field, human glucose-6-phosphate dehydrogenase was found to display kinetics at 0°C  within okcat/KM within the same magnitude order than at 37°C, while any remaining C1-INH are less functional. This could explain, why shipment of HAE-patient plasma samples at 0–4°C is definitely counterproductive for KKS analysis [Results of the present study demonstrate that PKa and FXIIa were active at 0°C with a analysis . Our obsanalysis .in vitro KKS reconstitution differed from MM kinetics, both initial burst and slower end reaction were also observed ex vivo in DS-activated plasma investigations, involving inhibitors and others proteases. In vivo KKS activation, in both physiological and pathological conditions (e.g. HAE) [Even in the absence of explanation of the mechanism by which .g. HAE) , is more.g. HAE) .Developing assay able to distinguish between HAE with normal C1-INH and histamine-mediated angioedema is challenging. To this end, plasma activation by DS has been investigated using submaximal doses of the KKS activator in a 30-min incubation at 37°C . Using aex vivo investigation of KKS in samples from HAE patients and from other inflammatory conditions.Kinetics of nHK cleavage by PKa and of PK activation by factor XII display parameters of the same magnitude at 0°C and at 37°C.in vitro KKS activation and plasma activation by DS show a fast-initial rate and a slow late rate.Kinetics of in vitro KKS activation.MM simulation does not fit with


Separately, Src homology 2 (SH2) domains mediate protein–protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr IQGAP1 is a scaffold protein with a multidomain architecture that allows for interactions with numerous diverse proteins ,3 to int1510  sequences [The SH2 domains of the non-receptor tyrosine kinases Abl1 and Abl2 were among those that docked to Tyrtive MET . Importaequences . Publishequences , SOCS3 [equences , ShcA [3equences , Src [39equences ; however1510 of IQGAP1 with non-phosphorylatable Ala enhances HGF-stimulated cell migration [To investigate whether IQGAP1 regulates Abl1 and Abl2 functions, we evaluated the phosphorylation of one of their substrates, namely the adaptor protein CrkL. IQGAP1 knockdown impairs basal phosphorylation of CrkL, implying that IQGAP1 promotes basal CrkL phosphorylation catalyzed by Abl. This observation could potentially result from the stimulation of Abl intrinsic kinase activity by IQGAP1. Although CrkL binding to IQGAP1 has not been documented, another possibility is that IQGAP1 scaffolds both the Abl kinases and CrkL to facilitate phosphorylation. In contrast to unstimulated cells, IQGAP1 knockdown increases HGF-induced CrkL phosphorylation, suggesting that IQGAP1 impairs HGF-stimulated Abl signaling. This result is likely to be a consequence of the reduction produced by IQGAP1 in MET activation stimulated by HGF that we observed in this study, which in turn reduces HGF-stimulated Abl activation. Together, our data demonstrate that IQGAP1 modulates both basal and growth factor-stimulated Abl signaling. The potential consequences of IQGAP1 on the biological functions of Abl proteins, namely cytoskeletal dynamics and DNA damage repair , remain igration . In lighigration , it is t1510 of IQGAP1 is catalyzed by the MET receptor [Phosphorylation of Tyrreceptor . While creceptor ,10,11,12Since the interaction of IQGAP1 with growth factor receptors often influences ligand-induced signaling . Taken tTo date, the roles of IQGAP1 in growth factor signaling have been generally documented to result from the constitutive scaffolding of receptors and signaling molecules by IQGAP1, independent of tyrosine phosphorylation. A well-characterized example is the role of IQGAP1 in EGFR-mediated activation of Raf kinase B. IQGAP1In conclusion, this work demonstrates the binding of IQGAP1 to MET, with functional consequences for both proteins. Moreover, it identifies for the first time that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins. Altogether, our study provides molecular insight into the regulation of signaling pathways and expands the mechanisms by which IQGAP1 scaffolds proteins to modulate intracellular signaling cascades.


Growing evidence indicates the pathogenic role of autoreactive IgE in autoimmune diseases. Incidence of autoimmune and allergic diseases in the industrialized countries is consistently icreasing, thus leading to concerted efforts to comprehend the regulation of IgE-mediated mechanisms. The first reports of a presence of IgE autoantibodies in patients with autoimmune diseases have been published a long time ago, and it is now recognized that self-reactive IgE can mediate inflammatory response in bullous pemhigoid, systemic lupus erythematosus, chronic urticaria, and atopic dermatitis. The advances in understanding the pathomechanisms of these disorders brought to a successful use of anti-IgE strategies in their management. The present review discusses the current state of knowledge on the IgE-mediated autoimmunity and anti-IgE treatment, and pave the way for further exploration of the subject. The importance of immunotolerance was recognized more than a half century ago and the groundbreaking studies were awarded with the Nobel Prize in Physiology or Medicine in 1960 for Macfarlane Burnet. Since then our understanding of immune system and the mechanisms that lead to autoimmunity has increased significantly. It is now known that the distinction between self and foreign is not absolute, and that it depends not only on genetic predisposition and biological sex but could be influenced by many environmental factors.Autoimmune diseases are not uncommon, with estimated prevalence of 7.6–9.4%  and predMany autoimmune diseases are characterized by the production of autoantibodies. Human B cells express five immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE. Generally, a classic B-cell response starts with the secretion of the IgM, followed by isotype switching and production of IgG, IgA, or IgE. Antibody (Ab) isotype switch is regulated by costimulatory signals provided by follicular T cells, cytokine milieu, and the nature of antigen (Ag) itself . The autOur knowledge of immunoglobulin E (IgE) has travelled a long way since the discovery of IgE by Kimishige Ishizaka and his wife and research partner Teruko Ishizaka in 1966 , to undeThere are two types of receptors for IgE: the high-affinity receptor FcεRI expressed as a classic αβγ2 tetrameric form by mast cells and basophils but also in a trimeric form by macrophages, dendritic cells (DCs), eosinophils, platelets and neutrophils, and the lower affinity receptor, CD23, present on various cells including mature B cells, activated T cells, Langerhans cells, plasma cells, monocytes, eosinophils, and epithelial cells . The FcεUntil today, the only anti-IgE therapy approved for clinical use is omalizumab, a recombinant humanized monoclonal antibody. Omalizumab, the humanized recombinant monoclonal antibody against IgE, is known to result in a marked reduction in serum-free IgE and the downregulation of IgE receptors on circulating basophils . Clinica+Tregs . The incidence of BP is 6 times higher in North America and Europe when compared to Asia . The patAtopic dermatitis (AD) is a chronic, non-infectious inflammatory disease of the skin of multifactoral etiology, including environmental factors, impaired skin barrier function, and immune system dysregulation . The patex vivo experiments confirmed functionality of anti-IL-24 IgE autoantibodies  guidelines, urticaria is a condition characterized by the spontaneos development of wheals , angioedema, or both, and is regarded as chronic if the above symptoms appearance due to known or unknown causes last more than 6 weeks . CSU is tibodies , and in tibodies . Neverthtibodies , and thetibodies . What’s tibodies . In a retibodies , but thetibodies .In a phase 2b core study in CSU patients , which wTwo more anti-IgE approaches were tested in CSU. The results of a randomized, double-blinded, placebo-controlled trial of quilizumab in adults with refractory CSU exhibited median reduction in serum IgE levels by approximately 30% over 20 week, but it did not cause clinically relevant improvements in CSU severity and activty scores . AnotherHyper-IgE syndromes (HIES) are rare primary immunodeficiencies characterized by elevated serum IgE, eczema and recurrent infections most commonly affecting skin and lungs, with the mean prevalence of less than 1:1000000 . The maiThe exact role of IgE in autoimmunity remains unclear, and it affects the decision on introducing the anti-IgE therapy. Fortunately, despite increased IgE levels in many autoimmune diseases the frequency of IgE mediated anaphylaxis is low . The pos


The Michaelis–Menten model of enzyme kinetic assumes the free ligand approximation, the steady-state approximation and the rapid equilibrium approximation. Analytical methods to model slow-binding inhibitors by the analysis of initial velocities have been developed but, due to their inherent complexity, they are seldom employed. In order to circumvent the complications that arise from the violation of the rapid equilibrium assumption, inhibition is commonly evaluated by pre-incubating the enzyme and the inhibitors so that, even for slow inhibitors, the binding equilibrium is established before the reaction is started. Here, we show that for long drug-target residence time inhibitors, the conventional analysis of initial velocities by the linear regression of double-reciprocal plots fails to provide a correct description of the inhibition mechanism. As a case study, the inhibition of acetylcholinesterase by galantamine, a drug approved for the symptomatic treatment of Alzheimer’s disease, is reported. For over 50 years, analysis based on the conventional steady-state model has overlooked the time-dependent nature of galantamine inhibition, leading to an erroneous assessment of the drug potency and, hence, to discrepancies between biochemical data and the pharmacological evidence. Re-examination of acetylcholinesterase inhibition by pre-steady state analysis of the reaction progress curves showed that the potency of galantamine has indeed been underestimated by a factor of ~100. For fast dissociating inhibitors (offk > 3 × 10−2 s−1), the inhibition is competitive (iu >>KicK). To evaluate the impact of an inhibitor’s residence time on the assessment of the steady-state inhibition parameters, synthetic kinetic data were generated using the software KinTek Explorer . Figure rsus [S] A,C,E howrsus [S] B, while rsus [S] F. In genoffk values in the 3 × 10−2–3 × 10−4 s−1 range, where the inhibition appears to be mixed-type and iuK decreases with the inhibitor residence time. Then it turns purely noncompetitive, with icK = iuK, for offk smaller than 3 × 10−4 s−1. The explanation of this apparent paradox is indeed trivial. When to start the reaction the substrate is added to the pre-incubated enzyme-inhibitor mixture, its binding causes a shift in the EI equilibrium according to the mass–action principle. For example, if the enzyme is incubated with an inhibitor concentration equal to iK, by definition at the equilibrium half of the total enzyme (e0) will be in the EI form, hence [EI] = [E] = 0.5 e0. If the reaction is started by adding the substrate in concentration equal to mK, a new equilibrium will be attained such that [EI] = [E] = [ES] = 0.33 e0. Hence, upon the reaction start, a partial dissociation of the EI complex takes place, which causes a corresponding apparent enzyme activation. In the presence of a competitive inhibitor the steady-state initial rate (vss) is given by:A gradual transition from competitive to noncompetitive occurs within 0 when the S is added to start the reaction, the portion of E available to interact with S is a fraction of the total enzyme equal to:If the enzyme is pre-incubated with the inhibitor, at time timaxV) is:measV) is:iuK = icK). For moderately slow EI dissociation rates, the measured initial velocities will be intermediate between ssv and measv, leading to an apparent mixed-type inhibition (icK < iuK < ~20 icK).Hence, if the EI dissociation is slow, the actual initial limiting velocity (offk larger than 1 s−1), the dissociation is not detected and it does not affect the measure of the initial velocities. On the contrary, if the dissociation occurs on a time scale similar to or larger than the typical dead time of the technique employed to monitor the reaction progress, it will be detected as an apparent enzyme activation. In this case, the recorded initial velocities underestimate the steady-state rates and the progress curves display an upward curvature resulting from the combination of EI dissociation and of substrate depletion.Because the monitoring of the reaction has a dead time that generally is not less than ~10 s (unless a stopped flow apparatus is used), for fast dissociating inhibitors . The time required for the recovery of the full enzyme activity is dependent only on offk and it can easily be calculated that the dissociation of ~95% of the bound inhibitor requires 3.5 times the residence time. Hence, once the reaction is started, a hypothetical inhibitor with offk of 10−2 s−1 would cause a slow reactivation that is completed in ~6 min , but in the case of the enzyme-initiated reactions their analysis would fail to detect any inhibition. Hence, in the latter case, the extent of the observed inhibition is largely dependent on the way the experiment is being carried out. Particularly relevant are the reagent concentrations and the portion of the progress curves that are integrated to detect the initial velocities. The faster the detection, the weaker the resulting apparent inhibition will be.Despite the non-compliance with the steady-state requirements these initial velocities are analyzed according to the Michaelis–Menten model. Depending on how the reactions are initiated, the result is either a large overestimation of the  20.7 nM C, while ± 2.7 nM D. It is ± 2.7 nM C. The reTcAChE inhibited by GAL was performed using the ENZO web application, implemented at www.enzo.cmm.ki.si  were constructed at substrate concentration in the 10–200 µM range. Data points are average values of three replicates. Four concentrations of inhibitors were selected for each assay: 50, 100, 200 and 400 nM for the enzyme-initiated reactions, and 5, 10, 20, 30 nM for the reaction started with the substrate. icK and iuK were determined by Dixon plot and Cornish–Bowden plot, respectively (see Reactions catalyzed by vely see .TcAChE, the hydrolysis of 35 µM acetylthiocholine by ~200 pM enzyme was monitored for 15 min. To assay the product inhibition by thiocholine-TNB, five consecutive reaction time courses were measured in the same 1 mL cuvette by adding 2 µL of a 20 mM fresh substrate solution once the previous reaction had come to completion so that four concentrations from 35 to 150 µM of the inhibitory thiocholine-TNB compound were assayed.Two distinct sets of progress curves were measured in triplicate either in the absence or in the presence of GAL. The analysis was performed using the ENZO web application. To evaluate the Michaelian parameters of the For the determination of GAL inhibition, the reactions with 200 pM enzyme and 50 µM acetylthiocholine were monitored for up to 33 min. Galantamine concentrations were 15, 30, 60, 120 and 180 nM.