scholarly journals On the Velocity of Enzymatic Reactions in Michaelis–Menten-Like Schemes (Ensemble and Single-Molecule Versions)

2020 ◽  
Vol 65 (5) ◽  
pp. 412 ◽  
Author(s):  
L. N. Christophorov
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Substrate Inhibition ◽  
Enzymatic Reaction ◽  
Enzymatic Reactions ◽  
Reaction Velocity ◽  
Conformational Regulation ◽  
Regulatory Properties

In searching non-standard ways of conformational regulation, various Michaelis–Menten-like schemes attract relentless attention, resulting in sometimes too sophisticated considerations. With the example of monomeric enzymes possessing an only binding site, we define the minimal schemes capable of bearing peculiar regulatory properties like “cooperativity” or substrate inhibition. The simplest ways of calculating the enzymatic reaction velocity are exemplified, either in the ensemble or single-molecule case.

Organophosphorus pesticides stress deranged kinetic values of a few midgut carbohydrases of naiad of Trithemis aurora (burm.) (Odonata: Libellulidae)

2016 ◽  
Vol 1 (02) ◽  
pp. 155-160
Author(s):  
Shekhar Chand
Keyword(s):  
Substrate Concentration ◽  
Initial Velocity ◽  
Ph Value ◽  
Organophosphorus Pesticides ◽  
Enzymatic Reaction ◽  
Organophosphorus Pesticide ◽  
Enzymatic Reactions ◽  
Reaction Velocity ◽  
Straight Line ◽  
Maximum Reaction

The 40 Hrs. treatment of last instar naiad of Trithemis aurora (Burm.) in Chlorphyriphos and Quinalphos pesticides concentrations (LC50 = 5.12 ×10-7 ppm and 7.6 × 10-8 ppm) has shown significant variations in the enzyme kinetic parameters and arrested the enzymatic activity in the midgut tissue of last instar naiad of T. aurora causing deleterious effect on various carbohydrases at standard temp. and pH value. The midgut amylase ( μ and β amylase) showed the change in the velocity of enzymatic reaction under LC50 conc. of chlorpyriphos. The data of initial velocity and substrate concentration were processed to achieve their reciprocal values. These values were plotted and a characteristic Lineweaver Burke straight line was observed from the graph and values of maximum reaction velocity (Vmax) and Michaelis Menten constants (km) were assessed. The present organophosphorus pesticide showed an inhibitory impact on midgut amylase reaction velocity. The double reciprocal plot of initial velocity and substrate concentration after exposing the enzyme under LC50 conc. of chlorphyriphos resulted in varied Vmax and Km. values. These carbohydrase on treatment with LC50 conc. of chlorphyriphos showed an inhibitory change in the reaction velocity. The 1/V and 1/S values were plotted to achieve a characteristic Lineweaver – Burke pattern of Vmax and km values obtained as 5.0 × 10-2 [M] and 2.0 under LC50 chlorphyriphos stress for α amylase. The km and Vmax values were obtained from 0.625 × 10-3 [M] to 1.25 × 10-2 [M] for various other midgut carbohydrases with Vmax value obtained from 0.28 to 5.0 under chlorpyriphos stress. The Quinalphos inhibited the enzymatic efficiencies of various carbohydrases severely and changed Km and Vmax values were found under the pesticidal stress and found as potent uncompetitive inhibitor for enzymes as values compared to the controlled enzymatic reactions by deranging the kinetic values. The Km values determined as on 1/V and 1/S basis found deranged from 1.66 × 10-3 [M] to 10 × 10-2 [M]. The Vmax values were found in a range of 0.41 to 3.3 under LC50 Quinalphos stress for midgut hydrolases. The analysis of enzymec kinetic values revealed the great inhibitory and deranged activities of various carbohydrases under both the pesticide constrain. The present toxicants were found to change the enzymatic velocity negatively. The LC50 concentrations of these toxicants were sufficient to inhibit the activity of present hydrolases as α and β amylase, α glucosidase, α galactosidase, β galactosidase, β frictosidase and α trehalase obtaining a meaningful Lineweaver – Burke line of plotted reciprocals of data of reaction velocity and substrate concentration.

scholarly journals 3D Kinetic Analysis: A Rapid, Model Independent, Method for Enzyme Kinetics

2021 ◽  
Author(s):  
Armina Abbasi ◽  
John T. Rodgers ◽  
Jeffrey P. Jones
Keyword(s):  
Enzyme Kinetics ◽  
Kinetic Models ◽  
Substrate Inhibition ◽  
Enzymatic Reaction ◽  
Time Dependent ◽  
General Strategy ◽  
Enzymatic Reactions ◽  
Maximal Velocity ◽  
Experimental Protocol ◽  
Over Time

Abstract In this work, we present the 3D Kinetics approach as a step forward in the field of enzyme kinetics. Normally in enzyme kinetics, it is first assumed that the kinetics will conform to the assumption of Michaelis and Menten and an experiment is conducted at various concentrations around the concentration that gives half-maximal velocity. Often, these experiments could be compromised by having too much substrate, nonlinear reaction over time, time-dependent or substrate inhibition, or several other kinetic models. Herein, we present a general strategy that will decrease the number of experiments required to develop an accurate representation of the kinetics of an enzymatic reaction. We show that with a single experimental protocol, we can fit a number of the most common kinetic models associated with enzyme-catalyzed reactions. Through this experiment, we introduce the effect of time on saturation curves by modeling the reaction velocity over time and across a set of substrate concentrations. Michaelis-Menten (MM) kinetics and other analytical solutions used to solve more complex kinetic models were introduced to the field of enzymology over a hundred years ago and have only marginally changed over the years with each analytical model requiring a different set of experiments and concentration ranges. Although this approach was necessary at the time, the computational power today makes any such simplifying and limiting efforts unnecessary and avoidable. In this study, we use a single experimental protocol and fit a number of different models to the resulting data. We present four different case studies to compare and contrast the outcomes of 3D Kinetics with MM analysis for different kinetic scenarios such as enzymatic reactions with linear kinetics, biphasic kinetics, substrate inhibition, and time-dependent inhibition (TDI) to confirm the advantage of the 3D Kinetics method to the long-established MM analysis.

scholarly journals Biological Nanopores: Engineering on Demand

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Ana Crnković ◽  
Marija Srnko ◽  
Gregor Anderluh
Keyword(s):  
Molecular Biology ◽  
Single Molecule ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Natural Sources ◽  
On Demand ◽  
Label Free Detection ◽  
Long Read ◽  
Inorganic Molecules ◽  
Standard Molecular Biology

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

scholarly journals Crystal Structures of Human Δ4-3-Ketosteroid 5β-Reductase (AKR1D1) Reveal the Presence of an Alternative Binding Site Responsible for Substrate Inhibition

Biochemistry ◽  
2009 ◽  
Vol 48 (5) ◽  
pp. 1144-1144
Author(s):  
Frédérick Faucher ◽  
Line Cantin ◽  
Van Luu-The ◽  
Fernand Labrie ◽  
Rock Breton
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Substrate Inhibition

scholarly journals Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis

2018 ◽  
Vol 115 (45) ◽  
pp. 11525-11530 ◽  
Author(s):  
Marcelo E. Guerin ◽  
Guillaume Stirnemann ◽  
David Giganti
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Conformational Sampling ◽  
Enzymatic Reactions ◽  
Sequence Specificity ◽  
Proteomics Data ◽  
Conformational Entropy ◽  
Substrate Peptide ◽  
The Impact

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.

scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

scholarly journals Hierarchical coupling between ATP hydrolysis and Hsp90’s client binding site

2020 ◽  
Author(s):  
Steffen Wolf ◽  
Benedikt Sohmen ◽  
Björn Hellenkamp ◽  
Johann Thurn ◽  
Gerhard Stock ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Information Transfer ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Substrate Binding ◽  
Signal Propagation ◽  
Nonequilibrium Molecular Dynamics ◽  
Time Resolved ◽  
Substrate Binding Site

I.ABSTRACTSeveral indicators for a signal propagation from a binding site to a distant functional site have been found in the Hsp90 dimer. Here we determined a time-resolved pathway from ATP hydrolysis to changes in a distant folding substrate binding site. This was possible by combining single-molecule fluorescence-based methods with extensive atomistic nonequilibrium molecular dynamics simulations. We find that hydrolysis of one ATP effects a structural asymmetry in the full Hsp90 dimer that leads to the collapse of a central folding substrate binding site. Arg380 is the major mediator in transferring structural information from the nucleotide to the substrate binding site. This allosteric process occurs via hierarchical dynamics that involve timescales from picoto milliseconds and length scales from Ångstroms to several nanometers. We presume that similar hierarchical mechanisms are fundamental for information transfer through many other proteins.

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

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