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Study on Enzyme Kinetics for Biochemistry

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Macromolecular Chemistry".

Deadline for manuscript submissions: closed (28 February 2022) | Viewed by 34699

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Dr. Marko Goliĉnik

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Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
Interests: enzyme kinetics; enzyme characterization; enzyme data documentation; numerical data analysis; enzyme high-throughput screening
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Special Issue Information

Dear colleagues,

It is an honour and great pleasure to invite you to contribute original research or review papers to this Special Issue of Molecules titled “Study on Enzyme Kinetics for Biochemistry”. Historically, enzyme kinetics have played an important role in biochemistry since the early discoveries of Henri, Michaelis and Menten, but today, enzyme kinetics also significantly complement omics data.

Therefore, we see the main goal of this Special Issue as sharing modern findings and results from this field among scientists. We will particularly be pleased to accept and review manuscripts that cover the following topics:

The practical aspects of measuring enzyme kinetics data;
Steady-state and pre-steady-state enzyme kinetics;
The numerical aspects of enzyme kinetics data analysis;
The mathematical modelling of regulatory enzyme processes;
Experimental factors, inhibitors and isotopes influencing enzyme kinetics;
Single-molecule and substrate-channelling enzyme kinetics.

Dr. Marko Goliĉnik
Guest Editor

Manuscript Submission Information

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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. Molecules is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

Enzyme catalysis
Kinetics of enzyme-catalyzed reactions
Inhibition of enzyme-catalyzed reactions
Regulation of enzyme-catalyzed reactions
Mathematical modelling and numerical enzymology

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Published Papers (10 papers)

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Research

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22 pages, 4830 KiB

Open AccessArticle

The Relationship between the IC₅₀ Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

by Pablo Garcia-Molina, Francisco Garcia-Molina, Jose Antonio Teruel-Puche, Jose Neptuno Rodriguez-Lopez, Francisco Garcia-Canovas and Jose Luis Muñoz-Muñoz

Molecules 2022, 27(10), 3141; https://doi.org/10.3390/molecules27103141 - 13 May 2022

Cited by 11 | Viewed by 4973

Abstract

Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (

i_{D}

), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of

i_{D}

with respect to the initial inhibitor concentration

{[I]}_{0}

allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC₅₀), thus avoiding making interpolations between different values of

i_{D}

. The analytical expression of the IC₅₀, for the different types of inhibition, are related to the apparent inhibition constant (

K_{I}^{app})

. Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC₅₀, the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC₅₀ value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in

i_{D}

and IC₅₀ with substrate concentration. The dependence of IC₅₀ on the substrate concentration allows us to distinguish between non-competitive inhibition (

i_{D}

does not depend on

{[D]}_{0}

) and the rest. In the case of competitive inhibition, this dependence of

i_{D}

{[D]}_{0}

leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of

K_{I_{1}}^{app}

is carried out from the IC₅₀ expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of

i_{D}

vs. n, with

n = {[D]}_{0} / K_{m}^{D}

, allows us to distinguish between them. A hyperbolic

i_{D}

vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of

K_{I_{2}}^{app}

is immediate from the IC₅₀ value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox),

K_{I_{1}}^{app}

and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD),

K_{I_{2}}^{app}

are obtained from the dependence of

i_{D}

vs. n, and the results obtained must comply with the IC₅₀ value. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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17 pages, 2494 KiB

Open AccessArticle

The Removal of Time–Concentration Data Points from Progress Curves Improves the Determination of K_m: The Example of Paraoxonase 1

by Boštjan Petrič, Marko Goličnik and Aljoša Bavec

Molecules 2022, 27(4), 1306; https://doi.org/10.3390/molecules27041306 - 15 Feb 2022

Cited by 5 | Viewed by 2622

Abstract

Several approaches for determining an enzyme’s kinetic parameter K_m (Michaelis constant) from progress curves have been developed in recent decades. In the present article, we compare different approaches on a set of experimental measurements of lactonase activity of paraoxonase 1 (PON1): (1) a differential-equation-based Michaelis–Menten (MM) reaction model in the program Dynafit; (2) an integrated MM rate equation, based on an approximation of the Lambert W function, in the program GraphPad Prism; (3) various techniques based on initial rates; and (4) the novel program “iFIT”, based on a method that removes data points outside the area of maximum curvature from the progress curve, before analysis with the integrated MM rate equation. We concluded that the integrated MM rate equation alone does not determine kinetic parameters precisely enough; however, when coupled with a method that removes data points (e.g., iFIT), it is highly precise. The results of iFIT are comparable to the results of Dynafit and outperform those of the approach with initial rates or with fitting the entire progress curve in GraphPad Prism; however, iFIT is simpler to use and does not require inputting a reaction mechanism. Removing unnecessary points from progress curves and focusing on the area around the maximum curvature is highly advised for all researchers determining K_m values from progress curves. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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21 pages, 2570 KiB

Open AccessArticle

The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses

by Clive R. Bagshaw, Jendrik Hentschel and Michael D. Stone

Molecules 2021, 26(24), 7532; https://doi.org/10.3390/molecules26247532 - 13 Dec 2021

Cited by 2 | Viewed by 2439

Abstract

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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14 pages, 1822 KiB

Open AccessArticle

Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies

by Alessandra Piccirilli, Emanuele Criscuolo, Fabrizia Brisdelli, Paola Sandra Mercuri, Sabrina Cherubini, Maria Laura De Sciscio, Mauro Maccarrone, Moreno Galleni, Gianfranco Amicosante and Mariagrazia Perilli

Molecules 2021, 26(18), 5489; https://doi.org/10.3390/molecules26185489 - 9 Sep 2021

Cited by 5 | Viewed by 1973

Abstract

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k_cat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of T_mB and T_mD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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19 pages, 4458 KiB

Open AccessArticle

The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

by Alina K. Bakunova, Alena Yu. Nikolaeva, Tatiana V. Rakitina, Tatiana Y. Isaikina, Maria G. Khrenova, Konstantin M. Boyko, Vladimir O. Popov and Ekaterina Yu. Bezsudnova

Molecules 2021, 26(16), 5053; https://doi.org/10.3390/molecules26165053 - 20 Aug 2021

Cited by 17 | Viewed by 3578

Abstract

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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12 pages, 5124 KiB

Open AccessArticle

The Interaction of Human Glutathione Transferase GSTA1-1 with Reactive Dyes

by Mohammed Hamed Alqarni, Ahmed Ibrahim Foudah, Magdy Mohamed Muharram and Nikolaos E. Labrou

Molecules 2021, 26(8), 2399; https://doi.org/10.3390/molecules26082399 - 20 Apr 2021

Cited by 5 | Viewed by 2682

Abstract

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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13 pages, 3985 KiB

Open AccessArticle

Boronic Acids as Prospective Inhibitors of Metallo-β-Lactamases: Efficient Chemical Reaction in the Enzymatic Active Site Revealed by Molecular Modeling

by Alexandra V. Krivitskaya and Maria G. Khrenova

Molecules 2021, 26(7), 2026; https://doi.org/10.3390/molecules26072026 - 2 Apr 2021

Cited by 12 | Viewed by 3078

Abstract

Boronic acids are prospective compounds in inhibition of metallo-β-lactamases as they form covalent adducts with the catalytic hydroxide anion in the enzymatic active site upon binding. We compare this chemical reaction in the active site of the New Delhi metallo-β-lactamase (NDM-1) with the hydrolysis of the antibacterial drug imipenem. The nucleophilic attack occurs with the energy barrier of 14 kcal/mol for imipenem and simultaneously upon binding a boronic acid inhibitor. A boron atom of an inhibitor exhibits stronger electrophilic properties than the carbonyl carbon atom of imipenem in a solution that is quantified by atomic Fukui indices. Upon forming the prereaction complex between NDM-1 and inhibitor, the lone electron pair of the nucleophile interacts with the vacant p-orbital of boron that facilitates the chemical reaction. We analyze a set of boronic acid compounds with the benzo[b]thiophene core complexed with the NDM-1 and propose quantitative structure-sroperty relationship (QSPR) equations that can predict IC50 values from the calculated descriptors of electron density. These relations are applied to classify other boronic acids with the same core found in the database of chemical compounds, PubChem, and proposed ourselves. We demonstrate that the IC50 values for all considered benzo[b]thiophene-containing boronic acid inhibitors are 30–70 μM. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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13 pages, 9723 KiB

Open AccessArticle

Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis

by Mohammed Hamed Alqarni, Ahmed Ibrahim Foudah, Magdy Mohamed Muharram, Haritium Budurian and Nikolaos E. Labrou

Molecules 2021, 26(5), 1222; https://doi.org/10.3390/molecules26051222 - 25 Feb 2021

Cited by 3 | Viewed by 2077

Abstract

The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD⁺. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (K_D = 0.66 ± 0.15 mM, first order maximum rate constant k₃ = 0.198 ± 0.06 min⁻¹). NAD⁺ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD⁺ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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Review

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18 pages, 445 KiB

Open AccessReview

Parameter Reliability and Understanding Enzyme Function

by Andrew G. McDonald and Keith F. Tipton

Molecules 2022, 27(1), 263; https://doi.org/10.3390/molecules27010263 - 1 Jan 2022

Cited by 18 | Viewed by 5079

Abstract

Knowledge of the Michaelis–Menten parameters and their meaning in different circumstances is an essential prerequisite to understanding enzyme function and behaviour. The published literature contains an abundance of values reported for many enzymes. The problem concerns assessing the appropriateness and validity of such material for the purpose to which it is to be applied. This review considers the evaluation of such data with particular emphasis on the assessment of its fitness for purpose. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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13 pages, 3476 KiB

Open AccessReview

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities

by Alexander A. Baykov, Viktor A. Anashkin and Anssi M. Malinen

Molecules 2021, 26(8), 2356; https://doi.org/10.3390/molecules26082356 - 18 Apr 2021

Cited by 10 | Viewed by 3707

Abstract

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP_i) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP_i as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP_i synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP_i hydrolysis, (b) continuous and fixed-time measurements of PP_i synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium’s composition, whose components are involved in multiple equilibria. Full article

(This article belongs to the Special Issue Study on Enzyme Kinetics for Biochemistry)

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