scholarly journals Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

2020 ◽  
Author(s):  
Piia Kokkonen ◽  
Andy Beier ◽  
Stanislav Mazurenko ◽  
Jiri Damborsky ◽  
David Bednar ◽  
...  
Keyword(s):  
Single Point ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Single Point Mutation ◽  
Substrate Complex ◽  
Enzyme Product ◽  
Enzyme Substrate ◽  
Markov State Models ◽  
State Models ◽  
Dynamics Simulations

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

scholarly journals Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

2020 ◽  
Author(s):  
Piia Kokkonen ◽  
Andy Beier ◽  
Stanislav Mazurenko ◽  
Jiri Damborsky ◽  
David Bednar ◽  
...  
Keyword(s):  
Single Point ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Single Point Mutation ◽  
Substrate Complex ◽  
Enzyme Product ◽  
Enzyme Substrate ◽  
Markov State Models ◽  
State Models ◽  
Dynamics Simulations

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

scholarly journals Assessing the capability ofin silicomutation protocols for predicting the finite temperature conformation of amino acids

2018 ◽  
Vol 20 (40) ◽  
pp. 25901-25909 ◽  
Author(s):  
Rodrigo Ochoa ◽  
Miguel A. Soler ◽  
Alessandro Laio ◽  
Pilar Cossio
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Molecular Dynamics Simulations ◽  
Point Mutation ◽  
Finite Temperature ◽  
Single Point ◽  
Single Point Mutation ◽  
Equilibrium Configurations ◽  
Dynamics Simulations

Single-point mutation protocols based on backbone-dependent rotamer libraries show the best performance in predicting equilibrium configurations from molecular dynamics simulations.

Properties of Chlorophyllase from Capsicum annuum L. Fruits

2001 ◽  
Vol 56 (11-12) ◽  
pp. 1015-1021 ◽  
Author(s):  
Dámaso Hornero-Méndez ◽  
María Isabel Mínguez-Mosquera
Keyword(s):  
Substrate Specificity ◽  
Capsicum Annuum ◽  
Mechanism Of Action ◽  
Substrate Recognition ◽  
Substrate Inhibition ◽  
Inhibitory Effect ◽  
Hplc Separation ◽  
Substrate Complex ◽  
Enzyme Substrate

Abstract The in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hy­drolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The enzym e showed an optimum of activity at pH 8.5 and 50 °C. Substrate specificity was studied for chlorophyll (Chi) a, Chi b, pheophytin (Phe) a and Phe b, with Km values of 10.70, 4.04, 2.67 and 6.37 μᴍ respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 μᴍ. Chlorophyllase action on Chi a' and Chi b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-132 in the chloro­ phyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+, Co , Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Func­tional groups such as -SH and -S-S-seem ed to participate in the formation o f the enzyme-substrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and product, has been optimized.

scholarly journals The mechanism of RNA base fraying: Molecular dynamics simulations analyzed with core-set Markov state models

2019 ◽  
Vol 150 (15) ◽  
pp. 154123 ◽  
Author(s):  
Giovanni Pinamonti ◽  
Fabian Paul ◽  
Frank Noé ◽  
Alex Rodriguez ◽  
Giovanni Bussi
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Markov State ◽  
Markov State Models ◽  
Core Set ◽  
State Models ◽  
Dynamics Simulations

scholarly journals The histone H3 N-terminal tail: a computational analysis of the free energy landscape and kinetics

2015 ◽  
Vol 17 (20) ◽  
pp. 13689-13698 ◽  
Author(s):  
Yuqing Zheng ◽  
Qiang Cui
Keyword(s):  
Free Energy ◽  
Energy Landscape ◽  
Critical Role ◽  
Histone H3 ◽  
Free Energy Landscape ◽  
Chromatin Dynamics ◽  
Markov State Models ◽  
State Models ◽  
Dynamics Simulations ◽  
Kinetics Of

Extensive molecular dynamics simulations and Markov State models are used to characterize the free energy landscape and kinetics of the histone H3 N-terminal tail, which plays a critical role in regulating chromatin dynamics and gene activity.

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