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Life
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Novel and Innovative Methods for Measuring Enzyme Activity
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Novel and Innovative Methods for Measuring Enzyme Activity

A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Proteins and Proteomics".

Deadline for manuscript submissions: closed (13 May 2022) | Viewed by 11412

Special Issue Editors

Dr. Angela Tramonti

E-Mail Website
Guest Editor
Institute of Molecular Biology and Pathology, CNR c/o Department of Biochemical Sciences, Sapienza University of Rome P.le A. Moro, 5 00185 Roma, Italy
Interests: enzymology; pyridoxal 5’-phosphate-dependent enzymes; vitamin B6 metabolism; cancer metabolic reprogramming; bacterial transcriptional regulation
Prof. Dr. Roberto Contestabile

E-Mail Website
Guest Editor
Department of Biochemical Sciences, Sapienza Università di Roma, 00185 Rome, Italy
Interests: enzyme kinetics; vitamin B6 metabolism; pyridoxal 5'-phosphate-dependent enzymes; bacterial transcriptional regulation; neonatal epileptic encephalopathy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Enzymes control virtually any cellular process and are at the very centre of biological research. Therefore, the methods employed to measure enzyme activity play a pivotal importance in most laboratories and are used to investigate the function of enzymes in metabolic pathways, cellular and pathological processes, and to study their mechanism of action. In vitro assays are often used to characterize the catalytic and kinetic properties of enzymes, enzyme inhibition mechanisms, reaction mechanisms, and structure-function relationships. A variety of approaches are used to look at different aspects of enzyme catalysis, such as steady state, progress curve, transient kinetics, and relaxation measurements. Ex vivo and in vivo enzyme assays are of great value to analyse the actual functioning of enzymes in biological samples, so as to investigate differentiation and pathological processes, tissue distribution, subcellular localization etc., using a great variety of methods.

This Special Issue focuses on any in vitro, ex vivo, and in vivo novel or innovative methods developed to assay enzyme activity.

Dr. Angela Tramonti
Prof. Dr. Roberto Contestabile
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Life is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • enzyme assays
  • enzyme kinetics
  • in vivo, ex vivo, and in vitro assays

Published Papers (4 papers)

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Research

13 pages, 1921 KiB  
Article
A Novel Assay for Phosphoserine Phosphatase Exploiting Serine Acetyltransferase as the Coupling Enzyme
by Francesco Marchesani, Erika Zangelmi, Stefano Bruno, Stefano Bettati, Alessio Peracchi and Barbara Campanini
Life 2021, 11(6), 485; https://doi.org/10.3390/life11060485 - 26 May 2021
Cited by 6 | Viewed by 3674
Abstract
Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis—the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through [...] Read more.
Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis—the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman’s reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP. Full article
(This article belongs to the Special Issue Novel and Innovative Methods for Measuring Enzyme Activity)
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10 pages, 2612 KiB  
Article
A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase
by Angela Tramonti, Roberto Contestabile, Rita Florio, Caterina Nardella, Anna Barile and Martino L. Di Salvo
Life 2021, 11(5), 438; https://doi.org/10.3390/life11050438 - 14 May 2021
Cited by 3 | Viewed by 2786
Abstract
Cysteine sulfinic acid decarboxylase catalyzes the last step of taurine biosynthesis in mammals, and belongs to the fold type I superfamily of pyridoxal-5′-phosphate (PLP)-dependent enzymes. Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal tissues; it is highly present in [...] Read more.
Cysteine sulfinic acid decarboxylase catalyzes the last step of taurine biosynthesis in mammals, and belongs to the fold type I superfamily of pyridoxal-5′-phosphate (PLP)-dependent enzymes. Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal tissues; it is highly present in liver, kidney, muscle, and brain, and plays numerous biological and physiological roles. Despite the importance of taurine in human health, human cysteine sulfinic acid decarboxylase has been poorly characterized at the biochemical level, although its three-dimensional structure has been solved. In the present work, we have recombinantly expressed and purified human cysteine sulfinic acid decarboxylase, and applied a simple spectroscopic direct method based on circular dichroism to measure its enzymatic activity. This method gives a significant advantage in terms of simplicity and reduction of execution time with respect to previously used assays, and will facilitate future studies on the catalytic mechanism of the enzyme. We determined the kinetic constants using L-cysteine sulfinic acid as substrate, and also showed that human cysteine sulfinic acid decarboxylase is capable to catalyze the decarboxylation—besides its natural substrates L-cysteine sulfinic acid and L-cysteic acid—of L-aspartate and L-glutamate, although with much lower efficiency. Full article
(This article belongs to the Special Issue Novel and Innovative Methods for Measuring Enzyme Activity)
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9 pages, 1114 KiB  
Article
An Electrochemical Approach to Follow and Evaluate the Kinetic Catalysis of Ricin on hsDNA
by George Oliveira and José Maurício Schneedorf
Life 2021, 11(5), 405; https://doi.org/10.3390/life11050405 - 29 Apr 2021
Viewed by 1434
Abstract
International authorities classify the ricin toxin, present in castor seeds, as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin are considered the first actions for its risk assessment during a suspected exposure, parallel to the development [...] Read more.
International authorities classify the ricin toxin, present in castor seeds, as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin are considered the first actions for its risk assessment during a suspected exposure, parallel to the development of therapeutic and medical countermeasures. In this study, we report the kinetic analysis of electro-oxidation of adenine released from hsDNA by the catalytic action of ricin by square wave voltammetry. The results suggest that ricin-mediated adenine release exhibited an unusual kinetic profile, with a progress curve controlled by the accumulation of the product and the values of the kinetic constants of 46.6 µM for Km and 2000 min−1 for kcat, leading to a catalytic efficiency of 7.1 × 105 s−1 M−1. Full article
(This article belongs to the Special Issue Novel and Innovative Methods for Measuring Enzyme Activity)
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13 pages, 3504 KiB  
Article
Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts
by Federico Mantoni, Chiara Scribani Rossi, Alessandro Paiardini, Adele Di Matteo, Loredana Cappellacci, Riccardo Petrelli, Massimo Ricciutelli, Alessio Paone, Francesca Cutruzzolà, Giorgio Giardina and Serena Rinaldo
Life 2021, 11(1), 31; https://doi.org/10.3390/life11010031 - 06 Jan 2021
Cited by 4 | Viewed by 2537
Abstract
GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding [...] Read more.
GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies. Full article
(This article belongs to the Special Issue Novel and Innovative Methods for Measuring Enzyme Activity)
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