Active-site chemistry of hemerythrin: kinetic studies on the reduction of the met octamer form from Themiste zostericola with [Co(sep)]2+, [Co(sarCl2]2+, [Co(9-aneN3)2]2+ and [Cr(bpy)3]2+

1985 ◽  
Vol 24 (20) ◽  
pp. 3230-3234 ◽  
Author(s):  
Graeme D. Armstrong ◽  
T. Ramasami ◽  
A. Geoffrey Sykes
Keyword(s):  
Active Site ◽  
Kinetic Studies

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals Synthesis and In Vitro Screening of Novel Heterocyclic β-d-Gluco- and β-d-Galactoconjugates as Butyrylcholinesterase Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2833
Author(s):  
Krešimir Baumann ◽  
Lorena Kordić ◽  
Marko Močibob ◽  
Goran Šinko ◽  
Srđanka Tomić
Keyword(s):  
Active Site ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
In Vitro Screening ◽  
Sugar Moiety ◽  
Brain Barrier Permeability ◽  
The Central Nervous System ◽  
Key Residues ◽  
Micromolar Range

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood–brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

scholarly journals Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase

2020 ◽  
Vol 117 (10) ◽  
pp. 5280-5290 ◽  
Author(s):  
Tamara V. Tikhonova ◽  
Dimitry Y. Sorokin ◽  
Wilfred R. Hagen ◽  
Maria G. Khrenova ◽  
Gerard Muyzer ◽  
...  
Keyword(s):  
Active Site ◽  
Copper Ions ◽  
Soda Lakes ◽  
Three Dimensional ◽  
Kinetic Studies ◽  
Isosceles Triangle ◽  
Site Directed Mutagenesis ◽  
Paramagnetic Resonance ◽  
Mechanism Of Catalysis ◽  
Copper Center

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

Meizothrombin Is Unexpectedly Zymogen-Like: Its Slow Conversion to Proteinase Dominates Thrombin Production by Prothrombinase

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2215-2215
Author(s):  
Harlan Bradford ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Conflicts Of Interest ◽  
Dominant Role ◽  
Kinetic Studies ◽  
Product Formation ◽  
Slow Step ◽  
Stopped Flow ◽  
Zymogen Activation ◽  
Proteolytic Activation ◽  
Initial Cleavage

Abstract Abstract 2215 The proteolytic activation of prothrombin catalyzed by prothrombinase is a paradigm for zymogen activation resulting from ordered cleavage at multiple sites. Initial cleavage at Arg320 forms meizothrombin (mIIa). The associated conversion of zymogen to proteinase is instrumental in facilitating further processing at Arg271 to yield thrombin. A full kinetic explanation for this process remains obscure and controversial because of the limitations of standard kinetic approaches. We now uncover novel facets of this reaction by rapid kinetic studies to approximate single catalytic turnover. Product formation was measured continuously by stopped flow using Dansyl arginine (3-ethyl-1,5-pentanediyl) amide (DAPA) or discontinuously using rapid quenching and analysis by SDS-PAGE or peptidyl substrate cleavage following rapid mixing of preformed prothrombinase (0.3 μM) with prothrombin (0.3 μM). Prothrombin cleavage, assessed discontinuously, was essentially complete within 0.2 seconds. The results indicated initial cleavage at Arg320 to form mIIa followed by subsequent cleavage at Arg271 to produce thrombin. However, product formation measured continuously with DAPA, yielded a pronounced lag and proceeded ∼20-30-fold more slowly. The intermediate, mIIa, which is expected to bind DAPA was invisible to the continuous measurements. Analysis was further simplified using a recombinant prothrombin variant, which is exclusively cleaved at Arg320 to produce mIIa and not processed further. Continuous detection of proteinase formation by stopped flow proceeded ∼30-fold more slowly than cleavage at Arg320 measured discontinuously. These findings indicate that rapid cleavage at the Arg320 site is followed by an unexpectedly slow reaction (t½ ∼ 0.5–1 s) in which the cleaved product matures to form a competent active site. This conclusion was further tested employing stable mIIa prepared by the action of Ecarin on recombinant prothrombin variants that were not degraded further even without occluding the active site. Stopped flow kinetic studies for the binding of DAPA to these variants revealed markedly biphasic traces. The data could be globally analyzed according to a two step mechanism with an initial slow equilibrium between zymogen-like and proteinase forms in which only the proteinase form can bind the active site ligand. The zymogen- like and proteinase forms were approximately equally populated and interconverted slowly (t½ ∼ 0.5 s). These findings independently corroborate the conclusions from the kinetic studies of prothrombin cleavage. Accordingly, inclusion of this slow step could explain profiles of prothrombin depletion, transient formation of mIIa and the final appearance of thrombin seen in the action of prothrombinase on prothrombin. Zymogen-like mIIa accumulates at much higher concentrations than would be predicted from knowledge of the kinetics of the individual cleavage steps because its slow maturation to proteinase is required for further cleavage at Arg271. Thus, the rate-limiting maturation of mIIa to proteinase plays a dominant role in regulating thrombin formation. Furthermore, while mIIa is a poor catalyst for many of substrates of thrombin, it retains the ability to bind thrombomodulin and function in the anticoagulant pathway. As a result of its unexpectedly zymogen-like character and slow conversion to proteinase, mIIa produced as an intermediate by prothrombinase would be resistant to inhibition in plasma and thereby potentially be dispersed by flowing blood to exert its selectively anticoagulant functions distant from its site of production. Disclosures: No relevant conflicts of interest to declare.

Spectroscopic and kinetic studies of the active site of anti-trinitrophenyl antibodies

1977 ◽  
Vol 14 (3) ◽  
pp. 227-229 ◽  
Author(s):  
Ronald P. Taylor ◽  
Richard L. Riley ◽  
Daniel J. Weber
Keyword(s):  
Active Site ◽  
Kinetic Studies

scholarly journals Kinetic Studies on Substrate Specificity and Active Site of .BETA.-D-Glucosidase F1 from Streptomyces sp.

2002 ◽  
Vol 49 (3) ◽  
pp. 265-272 ◽  
Author(s):  
Kenji Fukuda ◽  
Kou Shirakawa ◽  
Haruhide Mori ◽  
Masayuki Okuyama ◽  
Atsuo Kimura ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Kinetic Studies ◽  
Streptomyces Sp
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