scholarly journals Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation

1995 ◽  
Vol 308 (1) ◽  
pp. 327-333 ◽  
Author(s):  
P J Meikle ◽  
A M Whittle ◽  
J J Hopwood
Keyword(s):  
Ternary Complex ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Random Order ◽  
Lysosomal Membrane ◽  
Complex Mechanism ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Inhibition Studies

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

scholarly journals The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806

1974 ◽  
Vol 143 (3) ◽  
pp. 511-520 ◽  
Author(s):  
U. C. Knopf
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Nucleoside Triphosphate ◽  
Subunit Structure ◽  
Bacterial Cells ◽  
Protein Subunit ◽  
Ph Optimum ◽  
Protamine Sulphate

The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH4)2SO4, protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as β′, β, χ1 and α (mol.wts. 160000, 150000, 98000, and 41000±10% respectively). χ1 is the temporary designation for a protein subunit, which might have the same functions as the σ subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41±1°C and a pH optimum of 8.2±0.1. Mg2+ and Mn2+ are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates.

scholarly journals Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes

2004 ◽  
Vol 383 (3) ◽  
pp. 517-527 ◽  
Author(s):  
Dinesh A. NAGEGOWDA ◽  
Thomas J. BACH ◽  
Mee-Len CHYE
Keyword(s):  
Brassica Juncea ◽  
Mevalonate Pathway ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Site Directed Mutagenesis ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Double Mutation ◽  
Calculated Mass

3-Hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 °C, with an energy of activation of 62.5 J·mol−1. Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6–BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6–BjHMGS1 has an apparent Km-acetyl-CoA of 43 μM and a Vmax of 0.47 μmol·mg−1·min−1, and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased Vmax, indicating some involvement of these residues in catalytic capacity. Unlike His6–BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6–BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

scholarly journals Comparison of prohormone-processing activities in islet microsomes and secretory granules: evidence for distinct converting enzymes for separate islet prosomatostatins.

1984 ◽  
Vol 99 (2) ◽  
pp. 578-587 ◽  
Author(s):  
B D Noe ◽  
G Debo ◽  
J Spiess
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Golgi Complex ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Differential Distribution ◽  
Ph Optimum ◽  
Prohormone Processing

In previous work we have examined the nature of converting enzymes for proinsulin, proglucagon, and prosomatostatin-I (PSS-I) in secretory granules isolated from anglerfish islets. The purpose of the present study was to extend the examination of precursor conversion to islet microsomes and to compare prohormone processing, including that of PSS-I and prosomatostatin-II (PSS-II), in islet secretory granules and microsomes. Microsomes (rough endoplasmic reticulum [RER] and Golgi complex) and secretory granules were prepared from anglerfish islets by differential and discontinuous density-gradient centrifugation. Microsomes were further fractionated into Golgi- and RER-enriched subfractions. Lysed secretory granule or microsome preparations were incubated in the presence of a mixture of radioactively labeled islet prohormones. Extracts of products generated were subjected to analysis by gel filtration and high-pressure liquid chromatography. Accuracy of product cleavage was monitored by comparing high-pressure liquid chromatography retention times from the radiolabeled in vitro conversion products with the retention times of labeled products from tissue extracts. All converting activity in microsomes was found to be similar to that in granules in that it had a pH optimum near pH 5 and was inhibited by p-chloromercuribenzoate. No significant differences in the converting activity of Golgi complex- and RER-enriched subfractions of microsomes was observed. The proinsulin, proglucagon, and PSS-II converting-enzymes, which were found in islet secretory granules, were also present and membrane-associated in islet microsomes. However, converting activity for PSS-I was displayed only in secretory granules. This suggests that two or more separate enzymes are involved in processing PSS-I and PSS-II, and that these enzymes have either differential distribution or differential activity in RER/Golgi complex and secretory granules. The demonstration of converting enzyme activity in islet microsomes supports the proposal that these enzymes may be synthesized at the RER and are internalized along with the prohormones.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Binding Mechanism and Structural Insights into the Identified Protein Target of Covid-19 with In-Vitro Effective Drug Ivermectin

2020 ◽  
Author(s):  
Parth Sarthi Sen Gupta ◽  
Satyaranjan Biswal ◽  
Saroj Kumar Panda ◽  
Abhik Kumar Ray ◽  
Malay Kumar Rana
Keyword(s):  
Ternary Complex ◽  
Bound State ◽  
Molecular Target ◽  
Cooperative Interaction ◽  
Effective Drug ◽  
Rna Dependent Rna Polymerase ◽  
Protein Target ◽  
Ternary Complex Formation ◽  
Structural Insights

<p>While an FDA approved drug Ivermectin was reported to dramatically reduce the cell line of SARS-CoV-2 by ~5000 folds within 48 hours, the precise mechanism of action and the COVID-19 molecular target involved in interaction with this in-vitro effective drug are unknown yet. Among 12 different COVID-19 targets studied here, the RNA dependent RNA polymerase (RdRp) with RNA and Helicase NCB site show the strongest affinity to Ivermectin amounting -10.4 kcal/mol and -9.6 kcal/mol, respectively. Molecular dynamics of corresponding protein-drug complexes reveals that the drug bound state of RdRp with RNA has better structural stability than the Helicase NCB site, with MM/PBSA free energy of -135.2 kJ/mol, almost twice that of Helicase (-76.6 kJ/mol). The selectivity of Ivermectin to RdRp is triggered by a cooperative interaction of RNA-RdRp by ternary complex formation. Identification of the target and its interaction profile with Ivermectin can lead to more powerful drug designs for COVID-19 and experimental exploration. </p>

Mycobacterial DNA GyrB Inhibitors: Ligand Based Pharmacophore Modelling and In Vitro Enzyme Inhibition Studies

2014 ◽  
Vol 14 (17) ◽  
pp. 1990-2005 ◽  
Author(s):  
Shalini Saxena ◽  
Janupally Renuka ◽  
Variam Jeankumar ◽  
Perumal Yogeeswari ◽  
Dharmarajan Sriram
Keyword(s):  
Enzyme Inhibition ◽  
Pharmacophore Modelling ◽  
Inhibition Studies

Coumaronochromone as antibacterial and carbonic anhydrase inhibitors from Aerva persica (Burm.f.) Merr.: experimental and first-principles approaches

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Muhammad Imran ◽  
Ahmad Irfan ◽  
Mohammed A. Assiri ◽  
Sajjad H. Sumrra ◽  
Muhammad Saleem ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
First Principles ◽  
Molecular Electrostatic Potential ◽  
Chemical Constituents ◽  
Bacterial Strains ◽  
Hirshfeld Analysis ◽  
Carbonic Anhydrase Inhibition ◽  
Inhibition Studies ◽  
Lowest Unoccupied Molecular Orbitals

AbstractThe Aerva plants are exceptionally rich in phytochemicals and possess therapeutics potential. Phytochemical screening shows that Aerva persica (Burm.f.) Merr. contains highest contents i.e., total phenolics, flavonoids, flavonols, tannins, alkaloids, carbohydrates, anthraquinones and glycosides. In-vitro antibacterial and enzymatic (carbonic anhydrase) inhibition studies on methanol extracts of A. persica indicated the presence of biological active constituents within chloroform soluble portions. Investigation in the pure constituents on the chloroform portions of A. persica accomplished by column chromatography, NMR and MS analysis. The bioguided isolation yields four chemical constituents of coumaronochromone family, namely aervin (1-4). These pure chemical entities (1-4) showed significant antibacterial activity in the range of 60.05–79.21 µg/ml against various bacterial strains using ampicillin and ciprofloxacin as standard drugs. The compounds 1-4 showed promising carbonic anhydrase inhibition with IC50 values of 19.01, 18.24, 18.65 and 12.92 µM, respectively, using standard inhibitor acetazolamide. First-principles calculations revealed comprehensive intramolecular charge transfer in the studied compounds 1-4. The spatial distribution of highest occupied and lowest unoccupied molecular orbitals, ionization potential, molecular electrostatic potential and Hirshfeld analysis revealed that these coumaronochromone compounds would be proficient biological active compounds. These pure constituents may be used as a new pharmacophore to treat leaukomia, epilepsy, glaucoma and cystic fibrosis.

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