Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme β-glucuronidase from Escherichia coli

Craig, Douglas B.; King, Steffany D.; Reinfelds, Gundars; Henderson, Anna; Wood, Tabitha E.

doi:10.1016/j.ijbiomac.2016.12.034

Cited by 8 publications

(3 citation statements)

References 28 publications

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“…The Winzor model [2] used suggested that the variance in mobility of the unmodified enzyme could be attributed to relatively small difference in size and effective charge. This is consistent with other studies [13][14][15]. The model gave plausible results for the prediction of changes in size, shape, and charge required to accompany the changes in observed mobilities of -galactosidase caused by labelling.…”

Section: Factors Affecting Electrophoretic Mobilitysupporting

confidence: 91%

“…Previous studies have suggested that the observed heterogeneity in electrophoretic mobility of individual enzyme molecules may result from relatively small differences in shape, size, and charge [13][14][15]. In order to assess this, -galactosidase was subjected to relatively small changes in its size, shape and charge through chemical modification.…”

Section: Resultsmentioning

confidence: 99%

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Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli beta-Galactosidase

Riehl,

Klassen,

Abas

et al. 2023

Preprint

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Escherichia coli β-galactosidase was labelled with 1 mM fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to standards of label after attachment of BSA in order to determine the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 minutes were measured using capillary electrophoresis. The average mobilities were found determined to be -1.99x10− 8 m2V− 1s− 1 ± 1.3x10− 9 m2V− 1s− 1 (N = 39), -2.16 x10− 8 m2V− 1s− 1 ± 1.9x10− 9 m2V− 1s− 1 (N = 46), and − 2.18 x10− 8 m2V− 1s− 1 ± 2.1x10− 9 (N = 39) respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.

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Section: Factors Affecting Electrophoretic Mobilitysupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli beta-Galactosidase

Riehl,

Klassen,

Abas

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…And these aspects are important and not dismissible: A little fluctuation in organisms and their contexts can lead to completely different outcomes as they manifest chaotic behaviors [52][53][54][55][56][57][58][59] . Even "copies" of the "same" enzyme have different catalytic properties [60][61][62] .…”

mentioning

confidence: 99%

Machine Learning Can Solve the Reproducibility Crisis by Supplanting Reductionist Statistics

Sadri¹

2022

Preprint

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Resolving the "replication crisis" is a top priority of the scientific community now. "Reproducibility" is claimed as a central tenet of science and the estimated economic and social burden is huge. Numerous proposals have been made. Still, there lacks not only an established solution but even an agreement on whether there exists a "crisis" or not. Here, by questioning the philosophical foundations of our study designs and analyses, I trace back the "crisis" to reductionist ontologies and methodologies ingrained in the modern statistical methods which have dominated biological, medical, psychological, and social sciences for a century. The crisis is not our inability to "reproduce results" but that we expect to be able to "reproduce results" despite neglecting almost all individual-level and contextual variables of complex processes.

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Molecular Insights Into β‐Glucuronidase Inhibition by Alhagi Graecorum Flavonoids: A Computational and Experimental Approach

Kamel,

Maodaa,

Al‐Shaebi

et al. 2024

ChemistryOpen

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In this study, we aimed to investigate the inhibitory mechanisms of β‐glucuronidase by flavonoids derived from Alhagi graecorum through both experimental and computational approaches. The activity of β‐glucuronidase was assessed using an in vitro enzyme inhibition assay, where myricetin and chrysoeriol were identified as potent inhibitors based on their low IC50 values. Kinetic studies were conducted to determine the inhibition type, revealing that both compounds exhibit noncompetitive inhibition of β‐glucuronidase‐catalyzed hydrolysis of PNPG. Molecular docking was employed to explore the binding affinities of the flavonoids, showing that myricetin formed the highest number of polar interactions with the enzyme. Additionally, molecular dynamics (MD) simulations were performed to evaluate the stability of the enzyme‐inhibitor complexes, demonstrating consistent trajectory behavior for both compounds, with significant energy stabilization. Interaction energy analyses highlighted the dominant role of electrostatic forces in myricetin′s inhibition mechanism, while Van der Waals forces were more prominent for chrysoeriol. The MM/PBSA method was used to calculate the binding free energies, with myricetin and chrysoeriol exhibiting the lowest values. Potential energy landscape analysis further revealed that β‐glucuronidase adopts a more closed conformation when bound to these inhibitors, limiting substrate access. These findings suggest that flavonoids from Alhagi graecorum hold promise for clinical applications, particularly in managing drug‐induced enteropathy.

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Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme β-glucuronidase from Escherichia coli

Cited by 8 publications

References 28 publications

Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli beta-Galactosidase

Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli beta-Galactosidase

Machine Learning Can Solve the Reproducibility Crisis by Supplanting Reductionist Statistics

Molecular Insights Into β‐Glucuronidase Inhibition by Alhagi Graecorum Flavonoids: A Computational and Experimental Approach

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