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Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Prolyl oligopeptidase is inactivated by inhibitors of both cysteine and serine proteinases. Early studies clearly established this enzyme to be a member of the family of serine proteinases. It is quite sensitive to inactivation by diisopropyl fluorophosphate (DFP) and radiolabelled DFP is incorporated into the enzyme in a 1:1 molar ratio60. The rat brain enzyme is inactivated by DFP at a rate constant greater than that for trypsin56. The titration curve of kcat/Km exhibits a pKa of 6.2 consistent with a serine proteinase57. It is of interest that the enzyme is not inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride48. This likely reflects inaccessibility of this bulky reagent to the active site. Chloromethyl ketones known to alkylate active site histidine residues of serine proteinases also inhibit prolyl oligopeptidase60.
Novel Reporter Probes for HSV1-tk Gene Expression
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Mian M. Alauddin, Juri G. Gelovani
Compound 15 was synthesized and radiolabeled with 18F recently (41), and only the nonradioactive compound was tested in vitro. The compound (40 µM) showed better binding affinity for HSV1-TK than acyclovir [ACV (acyclovir; (9-(2-hydroxyethoxymethyl)guanine)), 170 mM] and ganciclovir (GCV, 48 µM). Catalytic turnover constant (Kcat) of 15 (0.08/sec) was close to the Kcat values of ACV (0.10/Sec) and GCV (0.10/sec). This compound did not show any antiviral activity against HSV1 and HSV2. Further animal biodistrbution and imaging studies are necessary to assess the efficacy of this compound. Compound 16 also has not been tested for biological evaluations (42,43).
Modulation of Tumor Matrix by Components of the Plasminogen-Plasmin System
Published in Róza Ádány, Tumor Matrix Biology, 2017
Following scu-PA binding to the receptor, its conversion to the two-chained form by Pn is greatly facilitated, estimated to be 50-fold greater than the in vivo action of Pn.90 The receptor-bound tcu-PA also converts Pg to Pn at a greater rate.90 The second-order catalytic efficiency of u-PA in Pg activation is increased sixfold on the cell surface due to a decrease of the Km from 25 μM in the fluid phase to 0.7 μM in the presence of cells.90,92 The inhibition by both PAI-1 and PAI-2 is decreased by about 40% after u-PA is bound to its receptor.90–92 Mapping studies on cells showed that the u-PA, bound to its receptor, is localized at focal contacts of cells, where they facilitate proteolysis at the adhesion sites during cell movement.95,96 Further analysis revealed that phosphorylation of the u-PA occurs during transportation to the cell surface from the cytosol.97,98 The six tyrosine residues in the ATF are phosphorylated by the src pp60 endogenous protein kinase and serine residues by protein kinase C, respectively. Whether this focal contact with the cytoplasm may initiate any signal transduction is the subject of intense investigation at the time of this review. The phosphorylated u-PA has different enzymatic properties from the non-phosphorylated form. Its catalytic effect of Pg expressed as Kcat, was fivefold that of the nonphosphorylated form, but the Km was 70-fold, resulting in a tenfold lower catalytic efficiency. However, the inhibition by PAI-1 and PAI-2 was much less in the phosphorylated form, approximately 35-fold less than inhibition of the non-phosphorylated u-PA.
Linear and branched β-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria
Published in Gut Microbes, 2020
Ravindra Pal Singh, Sivasubramanian Rajarammohan, Raksha Thakur, Mohsin Hassan
Michaelis Menten parameters of each enzyme in terms of Km, Vmax and Kcat were determined using various concentrations of different substrates, such as laminarin, curdlan, lichenan, and lentinan. Different concentrations of these substrates, ranging from 0.001 to 20 mg/ml were used for kinetics parameters with suitable concentration of enzyme in 50 mM sodium – phosphate buffer (pH 7.0) at 37°C for 2 h of incubation. Afterward, reaction was stopped by incubating at 100°C for 5 minutes. Enzymatic assays with these substrates were determined with DNS assays, as above mentioned,44 and thin-layer chromatography (TLC). TLC analysis was carried out on Silica Gel 60 F254 (Merck) and generated mono- and oligo-saccharides were visualized by spraying TLC with 5% H2SO4 in ethanol, followed by charring. Kinetics parameters such as Km, Vmax, and Kcat were analyzed on GraphPad Prism software. Three independent tests were performed for each experiment.
Examination of sulfonamide-based inhibitors of MMP3 using the conditioned media of invasive glioma cells
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Alisha T. Poole, Christopher A. Sitko, Caitlin Le, Christian C. Naus, Bryan M. Hill, Eric A. C. Bushnell, Vincent C. Chen
The leucine-tryptophan (Leu-Trp) backbone, and AP-1 were synthesised. All synthesised compounds were analysed by mass spectrometry (Figure S2). The NFF-3 assay was used to determine the biological inhibition of MMP-3 activity for ilomastat and synthesised compounds, Leu-Trp, and AP-1 relative to untreated controls. NFF-3 is a substrate selective for MMP3 (kcat/Km = 218,000 s−1 M−1), and to a much lesser degree, MMP9 (kcat/Km = 10,000 s−1 M−1)54. Consistent with our MMP3 SDS-PAGE/westernblot datum, the conditioned medium of C6-Cx43 demonstrated robust fluorescence activity compared to low motility C6 cells and unconditioned DMEM18. Linear responses of MMP3 activity was robustly measured from 30 to 300 min. All compounds were tested at a concentration of 50 μM and 100 μM, and compared to determine MMP3 suppression. From Figure 4 concentration dependent inhibition was observed. Here it can be seen that for all compounds the 100 μM was better at inhibiting MMP-3 than the 50 μM concentration. This analysis also demonstrates Leu-Trp backbone can act as a competitive inhibitor. Consistent with this interpretation, as discussed above in the docking study, Leu-Trp was found to ligate the Zn2+ via the Trp and Leu backbone carbonyl O-atoms. Demonstrating the relative contributions of the ZBG in AP-1 and ilomastat, both compounds perform better than Leu-Trp at inhibiting MMP-3.
Acetyl-CoA carboxylase (ACC) as a therapeutic target for metabolic syndrome and recent developments in ACC1/2 inhibitors
Published in Expert Opinion on Investigational Drugs, 2019
Leyuan Chen, Yuqing Duan, Huiqiang Wei, Hongxin Ning, Changfen Bi, Ying Zhao, Yong Qin, Yiliang Li
Like many enzymes, ACC is regulated in both the mRNA and protein levels in the body (Figure 1). The expression of the ACC gene is regulated by several transcription factors, including sterol regulatory element binding proteins (SREBP1a and SREBP1c) and carbohydrate response element binding protein (ChREBP) [14–16]. Under high glucose diets, SREBP and ChREBP can be activated by the insulin pathway, further promoting the expression of the ACC gene (Figure 1). In human tissues, citrate, as a precursor of acetyl-CoA, can allosterically activate ACC. Steady-state kinetic studies have reported that citrate increases the kcat and kcat/KM of the substrate ATP and acetyl-CoA of human ACC2 [17]. Acute regulation of ACC is achieved by reversible phosphorylation. Phosphorylation of Ser117 of ACC1 and Ser222 of ACC2 by AMPK (AMP-activated protein kinase) changes the ACCs from the active form of a large linear polymer to the inactive form [18].