InterferENZY: A Web-Based Tool for Enzymatic Assay Validation and Standardized Kinetic Analysis. Issue 11 (28th May 2021)

• Record Type: Journal Article
• Authors: Pinto, Maria Filipa; Baici, Antonio; Pereira, Pedro José Barbosa; Macedo-Ribeiro, Sandra; Pastore, Annalisa; Rocha, Fernando; Martins, Pedro M.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 11(2021)
• Publication Date: 2021-05-28
• Subjects: LM linearization method; enzyme kinetics -- webserver -- data pollution -- linearization method -- reproducibility; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.07.025
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Enzymatic assays are widely employed to characterize important allosteric and enzyme modulation effects. The high sensitivity of these assays can represent a serious problem if the occurrence of experimental errors surreptitiously affects the reliability of enzyme kinetics results. We have addressed this problem and found that hidden assay interferences can be unveiled by the graphical representation of progress curves in modified reaction coordinates. To render this analysis accessible to users across all levels of expertise, we have developed a webserver, interferENZY, that allows (i) an unprecedented tight quality control of experimental data, (ii) the automated identification of small and major assay interferences, and (iii) the estimation of bias-free kinetic parameters. By eliminating the subjectivity factor in kinetic data reporting, interferENZY will contribute to solving the "reproducibility crisis" that currently challenges experimental molecular biology. The interferENZY webserver is freely available (no login required) at https://interferenzy.i3s.up.pt . Graphical abstract: Unlabelled Image Highlights: Enzyme kinetics are used to characterize enzyme activity and its modulation. Yet, results can be distorted by assay interferences and improper data analysis. interferENZY automatically detects hidden assay interferences in time-course data. For valid assays, interferENZY provides unbiased estimates of kinetic parameters. interferENZY can improve accuracy and reproducibility in enzymology data reporting.