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Ligand Binding in Enzyme Systems
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Ligand Binding in Enzyme Systems

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (15 February 2021) | Viewed by 25022

Special Issue Editors

Prof. Dr. Francesco Malatesta

E-Mail Website
Guest Editor
Department of Biochemical Sciences, Sapienza University of Rome, 00185 Rome, Italy
Interests: bioenergetics; biophysics; chemical biology; enzymology kinetics; molecular medicine; protein chemistry structural; biology
Special Issues, Collections and Topics in MDPI journals
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

From a chemical point of view, the primary event in most biological processes is a second-order reaction, i. e., the reversible binding of a ligand to a specific target (protein, receptor, nucleic acid, or enzyme). In general, the binding dynamics may involve induced-fit or conformational selection mechanisms. In the world of enzymes, ligand binding, other than substrate binding, offers additional levels of complexity, whereby the enzyme may be regulated by feedback, product, and allosteric positive and negative modulation or may acquire new enzymatic activities or novel biological functions, as in moonlighting enzymes.

This Special Issue concentrates on the role of ligand binding to enzymes at the onset of the perturbation triggered by the ligand, both from thermodynamic and kinetic viewpoints. Original research articles and sharp up-to-date reviews on these and related topics are welcome in this Special Issue.

Prof. Francesco Malatesta
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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. 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
  • ligand binding
  • allosteric enzymes
  • allostery
  • enzyme regulation
  • enzyme inhibition
  • moonlighting enzymes
  • steady-state kinetics
  • pre-steady-state kinetics
  • stopped-flow spectroscopy

Published Papers (7 papers)

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Research

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17 pages, 4211 KiB  
Article
Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity
by Pablo Maturana, María S. Orellana, Sixto M. Herrera, Ignacio Martínez, Maximiliano Figueroa, José Martínez-Oyanedel, Victor Castro-Fernandez and Elena Uribe
Int. J. Mol. Sci. 2021, 22(9), 4769; https://doi.org/10.3390/ijms22094769 - 30 Apr 2021
Cited by 7 | Viewed by 2915
Abstract
Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase [...] Read more.
Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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27 pages, 7176 KiB  
Article
Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties
by Giovanna De Simone, Alessandra di Masi, Alessandra Pesce, Martino Bolognesi, Chiara Ciaccio, Lorenzo Tognaccini, Giulietta Smulevich, Stefania Abbruzzetti, Cristiano Viappiani, Stefano Bruno, Sara Della Monaca, Donatella Pietraforte, Paola Fattibene, Massimo Coletta and Paolo Ascenzi
Int. J. Mol. Sci. 2021, 22(4), 1674; https://doi.org/10.3390/ijms22041674 - 7 Feb 2021
Cited by 10 | Viewed by 2323
Abstract
Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was [...] Read more.
Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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10 pages, 951 KiB  
Communication
NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins
by Giovanna De Simone, Alessandra di Masi, Chiara Ciaccio, Massimo Coletta and Paolo Ascenzi
Int. J. Mol. Sci. 2020, 21(24), 9395; https://doi.org/10.3390/ijms21249395 - 10 Dec 2020
Cited by 10 | Viewed by 1369
Abstract
Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The [...] Read more.
Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10−5 and 1.0 × 10−3 M), NO binding is lost in the mixing time (i.e., NOkon ≥ 1.0 × 106 M−1 s−1). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., klim) whose values increase linearly with [OH−]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH−-based catalysis. From the dependence of klim on [OH−], the values of the second-order rate constant kOH− for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 103 M−1 s−1 and 6.9 (±0.8) × 103 M−1 s−1, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO2 and another being tightly bound to the ferrous heme-Fe(II) atom. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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21 pages, 3896 KiB  
Article
Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90
by Sang Chul Shin, Ashraf K. El-Damasy, Ju Hyeon Lee, Seon Hee Seo, Ji Hyun Kim, Young Ho Seo, Yuri Lee, Ji Hoon Yu, Eun Kyoung Bang, Eunice EunKyeong Kim and Gyochang Keum
Int. J. Mol. Sci. 2020, 21(24), 9377; https://doi.org/10.3390/ijms21249377 - 9 Dec 2020
Cited by 7 | Viewed by 2455
Abstract
Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, [...] Read more.
Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N7/N9-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC50 values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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11 pages, 2470 KiB  
Article
Strand Displacement Activity of PrimPol
by Elizaveta O. Boldinova, Ekaterina A. Belousova, Diana I. Gagarinskaya, Ekaterina A. Maltseva, Svetlana N. Khodyreva, Olga I. Lavrik and Alena V. Makarova
Int. J. Mol. Sci. 2020, 21(23), 9027; https://doi.org/10.3390/ijms21239027 - 27 Nov 2020
Cited by 7 | Viewed by 2372
Abstract
Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and [...] Read more.
Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5′-DNA flaps generated by PrimPol in vitro. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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12 pages, 1386 KiB  
Article
Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex
by Elena Forte, Alessandro Giuffrè, Li-shar Huang, Edward A. Berry and Vitaliy B. Borisov
Int. J. Mol. Sci. 2020, 21(22), 8521; https://doi.org/10.3390/ijms21228521 - 12 Nov 2020
Cited by 9 | Viewed by 2052
Abstract
Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type [...] Read more.
Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)−1 min−1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)−1 min−1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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Review

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36 pages, 3801 KiB  
Review
Arginase as a Potential Biomarker of Disease Progression: A Molecular Imaging Perspective
by Gonçalo S. Clemente, Aren van Waarde, Inês F. Antunes, Alexander Dömling and Philip H. Elsinga
Int. J. Mol. Sci. 2020, 21(15), 5291; https://doi.org/10.3390/ijms21155291 - 25 Jul 2020
Cited by 61 | Viewed by 10759
Abstract
Arginase is a widely known enzyme of the urea cycle that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The action of arginase goes beyond the boundaries of hepatic ureogenic function, being widespread through most tissues. Two arginase isoforms coexist, the type [...] Read more.
Arginase is a widely known enzyme of the urea cycle that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The action of arginase goes beyond the boundaries of hepatic ureogenic function, being widespread through most tissues. Two arginase isoforms coexist, the type I (Arg1) predominantly expressed in the liver and the type II (Arg2) expressed throughout extrahepatic tissues. By producing L-ornithine while competing with nitric oxide synthase (NOS) for the same substrate (L-arginine), arginase can influence the endogenous levels of polyamines, proline, and NO•. Several pathophysiological processes may deregulate arginase/NOS balance, disturbing the homeostasis and functionality of the organism. Upregulated arginase expression is associated with several pathological processes that can range from cardiovascular, immune-mediated, and tumorigenic conditions to neurodegenerative disorders. Thus, arginase is a potential biomarker of disease progression and severity and has recently been the subject of research studies regarding the therapeutic efficacy of arginase inhibitors. This review gives a comprehensive overview of the pathophysiological role of arginase and the current state of development of arginase inhibitors, discussing the potential of arginase as a molecular imaging biomarker and stimulating the development of novel specific and high-affinity arginase imaging probes. Full article
(This article belongs to the Special Issue Ligand Binding in Enzyme Systems)
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