Ping-pong Mechanism Research Articles

Mechanistic and structural insights into EstS1 esterase: A potent broad-spectrum phthalate diester degrading enzyme.

Phthalate diesters are important pollutants and act as endocrine disruptors. While certain bacterial esterases have been identified for phthalate diesters degradation to monoesters, their structural and mechanistic characteristics remain largely unexplored. Here, we highlight the potential of the thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus DSM10332 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by combining biophysical and biochemical approaches along with high-resolution EstS1 crystal structures of the apo form and with bound substrates, products, and their analogs to elucidate its mechanism. The catalytic tunnel mediates entry and exit of the substrate and product, respectively. The centralized Ser-His-Asp triad performs catalysis by a bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Mutagenesis analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore EstS1 as a promising tool for advancing technologies aimed at phthalate diesters biodegradation.

Embryonic piRNAs target horizontally transferred vertebrate transposons in assassin bugs.

Piwi proteins and the associated Piwi-interacting RNAs (piRNAs) coordinate a surveillance system that protects the animal genome from DNA damage induced by transposable element (TE) mobilization. While the pathway has been described in detail in the fruit fly Drosophila melanogaster, much less is known in more basal insects. Rhodnius prolixus is an hemipteran insect and one of the major vectors of Chagas disease. Rhodnius acquired specific classes of horizontally transferred transposons (HTTs) by feeding on bats, opossums and squirrel monkeys, thus providing the opportunity to investigate the piRNA-base response against HTTs in this species. SmallRNA-Seq reads mapping to HTTs and resident transposable elements were quantified and checked for piRNA features like 1U a 10A biases, ping-pong and phasing signatures. Uniquely mapped piRNAs were used to identify piRNA clusters in Rhodnius' genome. RNA-Seq data was used to quantify transposon and Rp-PIWI genes expression levels and were validated by qRT-PCR. By analyzing the temporal dynamics of piRNA cluster expression and piRNA production during critical stages of Rhodnius development, we show that peak levels of ∼28nt long piRNAs correlate with reduced HTT and resident TE expression primarily during embryogenesis. Strikingly, while resident TEs piRNAs seem to engage in a typical ping-pong amplification mechanism, sense and antisense HTT piRNAs instead overlap by ∼20nt or do not display ping-pong signatures. Our data shed light on the biogenesis and functions of the piRNAs in Rhodnius prolixus and reveal that piRNAs, but not the siRNA pathway, responded to HTTs that were recently transferred from vertebrate tetrapods to a hematophagous insect of medical relevance.

Structural basis of the mechanism and inhibition of a human ceramide synthase.

Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery.

Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Ultrastable cobalt-based chainmail catalyst for degradation of emerging contaminants in water

Developing stable and efficient cobalt-based catalysts for degradation of emerging contaminants (ECs) in water is deemed as a practical strategy to avoid secondary pollution in advanced oxidation processes (AOPs). Herein, we developed a ultrastable cobalt-based chainmail catalysts (Co-NC900) using 2, 2’-bipyridine-assistant pyrolysis method. More than 96.6 % of imidacloprid (IMD) in water could be degraded within 5 min in the presence of Co-NC900 and peroxymonosulfate (PMS). Amazingly, Co-NC900 still give 95 % degradation efficacy for IMD after15 runs. And only 0.083 μg L−1 of Co could be detected in the reaction mixture after continuous using 7 d. Density Functional Theory (DFT) calculations demonstrated that Co-NC900 can efficiently activate PMS through unique ping-pong mechanism (C-N bond breaking and re-forming process), and further degrade ECs. This study presents a novel approach for the development of efficient and stable metal-based catalysts for the removal of ECs from water.

Crystal structures of NAD(P)H nitroreductases from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections andits tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen-insensitive nitroreductases (Kp-NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp-NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)-binding mode is provided. The structures with PDB codes 7tmf (Kp-NR1a), 7tmg (Kp-NR1b) and 8dor (Kp-NR2) were determined at resolutions of 1.97, 1.90 and 1.35 Å, respectively. The Kp-NR1a and Kp-NR1b structures adopt an αβ fold, in which four-stranded antiparallel β-sheets are surrounded by five helices. With domain swapping, the β-sheet was expanded with a β-strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173-Ala185: in Kp-NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp-NR1b. The FMN interactions within Kp-NR1/NR2 involve hydrogen-bond and π-stacking interactions. Kp-NR2 contains four-stranded antiparallel β-sheets surrounded by eight helices with two short helices and one β-sheet. Structural and sequence alignments show that Kp-NR1a/b and Kp-NR2 are homologs of the Escherichia coli oxygen-insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp-NR1a/b and Kp-NR2 may detoxify polynitroaromatic compounds and Kp-NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping-pong bi-bi mechanism, respectively.

Mechanism of ceramide synthase inhibition by fumonisin B1

Ceramide synthases (CerSs) play crucial roles in sphingolipid metabolism and have emerged as promising drug targets for metabolic diseases, cancers, and antifungal therapy. However, the therapeutic targeting of CerSs has been hindered by a limited understanding of their inhibition mechanisms by small molecules. Fumonisin B1 (FB1) has been extensively studied as a potent inhibitor of eukaryotic CerSs. In this study, we characterize the inhibition mechanism of FB1 on yeast CerS (yCerS) and determine the structures of both FB1-bound and N-acyl-FB1-bound yCerS. Through our structural analysis and the observation of N-acylation of FB1 by yCerS, we propose a potential ping-pong catalytic mechanism for FB1 N-acylation by yCerS. Lastly, we demonstrate that FB1 exhibits lower binding affinity for yCerS compared to the C26- coenzyme A (CoA) substrate, suggesting that the potent inhibitory effect of FB1 on yCerS may primarily result from the N-acyl-FB1 catalyzed by yCerS, rather than through direct binding of FB1.

Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.

Fraternal twins at work: Structures of PLD3/4 reveal mechanism for lysosomal nucleic acid breakdown

Phospholipase D (PLD) family proteins degrade phospholipids and nucleic acids. In the current issue of Structure, Yuan etal.1 report crystal structures of lysosomal PLD3 and PLD4 with and without a single-stranded DNA substrate. Their manuscript reveals a catalytic ping-pong mechanism and explains how disease-associated mutations compromise PLD3/4 function.

Mutational and structural studies of (βα)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism.

Methylene-tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one-carbon (C1) metabolisms. F420-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H4F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H4F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer-F420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride-transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl-tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5-iminium cation of methylene-tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out.

Catalytic Mechanism of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A.

The oxidation of Met to methionine sulfoxide (MetSO) by oxidants such as hydrogen peroxide, hypochlorite, or peroxynitrite has profound effects on protein function. This modification can be reversed by methionine sulfoxide reductases (msr). In the context of pathogen infection, the reduction of oxidized proteins gains significance due to microbial oxidative damage generated by the immune system. For example, Mycobacterium tuberculosis (Mt) utilizes msrs (MtmsrA and MtmsrB) as part of the repair response to the host-induced oxidative stress. The absence of these enzymes makes Mycobacteria prone to increased susceptibility to cell death, pointing them out as potential therapeutic targets. This study provides a detailed characterization of the catalytic mechanism of MtmsrA using a comprehensive approach, including experimental techniques and theoretical methodologies. Confirming a ping-pong type enzymatic mechanism, we elucidate the catalytic parameters for sulfoxide and thioredoxin substrates (kcat/KM = 2656 ± 525 M-1 s-1 and 1.7 ± 0.8 × 106 M-1 s-1, respectively). Notably, the entropic nature of the activation process thermodynamics, representing ∼85% of the activation free energy at room temperature, is underscored. Furthermore, the current study questions the plausibility of a sulfurane intermediate, which may be a transition-state-like structure, suggesting the involvement of a conserved histidine residue as an acid-base catalyst in the MetSO reduction mechanism. This mechanistic insight not only advances our understanding of Mt antioxidant enzymes but also holds implications for future drug discovery and biotechnological applications.

Exploring the Intricacies and Functionalities of Galactose Oxidase: Structural Nuances, Catalytic Behaviors, and Prospects in Bio-electrocatalysis

Galactose Oxidase, also known as GOase, is an enzyme found mostly in Fusarium graminearum, Dactylium dendroides, and Gibberella fujikuroi. GOase, containing copper, serves catalytic functions in oxidizing substrates and primary alcohols such as d-galactose, benzyl alcohol derivatives, and dihydroxyacetone. The catalytic property of galactose oxidase that differentiates it from other enzymes is its cofactor consisting of a Cu (II)-bound Cys-Tyr* radical. The cofactor is vital for enabling regioselective oxidation. The application of galactose oxidase (GOase) covers several fields such as enzymatic synthesis, biosensors development, and processes of diagnosis.
 Galactose oxidase (GOase) was discovered to have a crystallographic structure by X-ray diffraction, which revealed an active site containing copper ions displaying relatively square pyramidal geometry. There are three unique structural and functional domains of the enzyme GOase. The domains include Tyr495 and a covalent bond between Cys228 and Tyr272 serving as equatorial and axial ligands, respectively. The mechanism of catalysis covers three different oxidation states, which include the active state containing Cu (II)-radical, the intermediate state containing Cu (II)-tyrosine, and the Cu(I)-tyrosine state. The cycle of catalysis that has been posited comprises several phases which are: (i) substrate binding, (ii) the transfer of proton (iii) the transfer of hydrogen atom, and (iv) subsequent oxidation steps. These phases eventually yield the synthesis of aldehyde and hydrogen peroxide.
 Galactose oxidase’s (GOase) mechanism of catalysis has been studied thoroughly via extensive research focusing on explaining the ping-pong mechanism that occurs in both oxidative and reductive half-reactions. The processes of activating and reactivating galactose (GOase) involve the transfer of electrons in which horseradish peroxidase (HRP) serves as an activator. The electrochemical investigations provide evidence of the electrochemical activation and reactivation of GOase in the presence of mediators.
 This comprehensive review enhances the comprehension of the structural complexities, catalytic mechanisms, and bio-electrocatalytic potential of GOase, thereby establishing a basis for future investigations and developments in technology.

Kinetic analysis of the three-substrate reaction mechanism of an NRPS-independent siderophore (NIS) synthetase.

The biosynthesis of many bacterial siderophores employs a member of a family of ligases that have been defined as NRPS-independent siderophore (NIS) synthetases. These NIS synthetases use a molecule of ATP to produce an amide linkage between a carboxylate and an amine. Commonly used carboxylate substrates include citrate or α-ketoglutarate, or derivatives thereof, while the amines are often hydroxamate derivatives of lysine or ornithine, or their decarboxylated forms cadaverine and putrescine. Enzymes that employ three substrates to catalyze a reaction may proceed through alternate mechanisms. Some enzymes use sequential mechanisms in which all three substrates bind prior to any chemical steps. In such mechanisms, substrates can bind in a random, ordered, or mixed fashion. Alternately, other enzymes employ a ping-pong mechanism in which a chemical step occurs prior to the binding of all three substrates. Here we describe an enzyme assay that will distinguish among these different mechanisms for the NIS synthetase, using IucA, an enzyme involved in the production of aerobactin, as the model system.

Theoretical calculation on degradation mechanism of novel copolyesters under CALB enzyme

Poly(butylene succinate-co-furandicarboxylate) (PBSF) and poly(butylene adipate-co-furandicarboxylate) (PBAF) are novel furandicarboxylic acid-based biodegradable copolyesters with great potential to replace fossil-derived terephthalic acid-based copolyesters such as poly(butylene succinate-co-terephthalate) (PBST) and poly(butylene adipate-co-terephthalate) (PBAT). In this study, quantum chemistry techniques after molecular dynamics simulations are employed to investigate the degradation mechanism of PBSF and PBAF catalyzed by Candida antarctica lipase B (CALB). Computational analysis indicates that the catalytic reaction follows a four-step mechanism resembling the ping-pong bibi mechanism, with the initial two steps being acylation reactions and the subsequent two being hydrolysis reactions. Notably, the first step of the hydrolysis is identified as the rate-determining step. Moreover, by introducing single-point mutations to expand the substrate entrance tunnel, the catalytic distance of the first acylation step decreases. Additionally, energy barrier of the rate-determining step is decreased in the PBSF system by site-directed mutations on key residues increasing hydrophobicity of the enzyme's active site. This study unprecedently show the substrate binding pocket and hydrophobicity of the enzyme's active site have the potential to be engineered to enhance the degradation of copolyesters catalyzed by CALB.

A biosensor for D-2-hydroxyglutarate in frozen sections and intraoperative assessment of IDH mutation status

The oncometabolite D-2-hydroxyglutarate (D-2-HG) has emerged as a valuable biomarker in tumors with isocitrate dehydrogenase (IDH) mutations. Efficient detection methods are required and rapid intraoperative determination of D-2-HG remains a huge challenge. Herein, D-2-HG dehydrogenase from Achromobacter xylosoxidans (AX-D2HGDH) was found to have high substrate specificity. AX-D2HGDH dehydrogenizes D-2-HG and reduces flavin adenine dinucleotide (FAD) bound to the enzyme. Interestingly, the dye resazurin can be taken as another substrate to restore FAD. AX-D2HGDH thus catalyzes a bisubstrate and biproduct reaction: the dehydrogenation of D-2-HG to 2-ketoglutarate and simultaneous reduction of non-fluorescent resazurin to highly fluorescent resorufin. According to steady-state analysis, a ping-pong bi-bi mechanism has been concluded. The Km values for resazurin and D-2-HG were determined as 0.56 μM and 10.93 μM, respectively, suggesting high affinity to both substrates. On the basis, taking AX-D2HGDH and resazurin as recognition and fluorescence transducing element, a D-2-HG biosensor (HGAXR) has been constructed. HGAXR exhibits high sensitivity, accuracy and specificity for D-2-HG in different biological samples. With the aid of HGAXR and the matched low-cost palm-size detecting device, D-2-HG levels in frozen sections of resected brain tumor tissues can be measured in a direct, simple and accurate manner with a fast detection (1–3 min). As the technique of frozen section is familiar to surgeons and pathologists, HGAXR and the portable device can be easily integrated into the current workflow, having potential to provide rapid intraoperative pathology for IDH mutation status and guide decision-making during surgery.

Condition optimization and kinetic evaluation of Novozym 435-catalyzed synthesis of aroma pyridine esters

A new method for rapid synthesis of pyridine esters without stirring based on Novozym 435 catalysis had been reported, which was from pyridine methanols and vinyl esters. Based on single factor experiment, the optimization of the enzymatic transesterification was received by response surface methodology (RSM). The high yield of 82% was still reached when Novozym 435 had been recycled 12 times. Under optimal conditions, a total of 22 pyridine esters were synthesized. The kinetic model of Novozym 435-catalyzed transesterification of vinyl acetate with 3-pyridinemethanol was established by enzymatic assay. It was discovered that the reaction followed the Ping-Pong Bi-Bi mechanism, and 3-pyridinemethanol inhibited the reaction. Gas chromatography-mass spectrometry-olfactometry (GC–MS-O) was used to analyze the synthesized pyridine esters, of which pyridin-3-ylmethylhexanoate (3e), pyridin-3-ylmethylcinnamate (3i), and 2-(pyridin-2-yl)ethyl acetate (3 s) possessed strong and excellent aromas. These three flavor compounds (3e, 3i and 3 s) were found to have good stability at the specified temperature by thermogravimetric (TG) analysis.

Intrinsic peroxidase-like activity of dissolved black carbon released from biochar

Dissolved black carbon (DBC) is an important constituent of the natural organic carbon pool, influencing the global carbon cycling and the fate processes of many pollutants. In this work, we discovered that DBC released from biochar has intrinsic peroxidase-like activity. DBC samples were derived from four biomass stocks, including corn, peanut, rice, and sorghum straws. All DBC samples catalyze H2O2 decomposition into hydroxyl radicals, as determined by the electron paramagnetic resonance and the molecular probe. Similar to enzymes that exhibit saturation kinetics, the steady-state reaction rates follow the Michaelis−Menten equation. The peroxidase-like activity of DBC is controlled by the ping-pong mechanism, as suggested by parallel Lineweaver−Burk plots. Its activity increases with temperature from 10 to 80 °C and has an optimum at pH 5. The peroxidase-like activity of DBC is positively correlated with its aromaticity as aromatics can stabilize the reactive intermediates. The active sites in DBC also involve oxygen-containing groups, as inferred by increased activity after the chemical reduction of carbonyls. The peroxidase-like activity of DBC has significant implications for biogeochemical processing of carbon and potential health and ecological impacts of black carbon. It also highlights the need to advance the understanding of the occurrence and role of organic catalysts in natural systems.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N-acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

A fractional model of enzymatic competitive substrate inhibition with Ping-Pong mechanism

Enzymes are biodegradable catalysts naturally present in living organisms. Enzymes can accelerate biochemical reactions by reducing the activation energy, and they are not consumed during reaction processes. Numerous applications of enzymes have been developed in biotechnology, industry, medicine, pharmaceuticals, food processing, biofuels, and so on. In this study, we develop a mathematical model describing enzymatic reactions with a Ping-Pong mechanism and competitive substrate inhibition. In order to obtain insights into the model behaviors, we use Python software to obtain numerical solutions for the model. Some discussions on the numerical results is provided. Finally, we briefly discuss a potential application of the model and some future work.

Improvements in the Modeling and Kinetics Processes of the Enzymatic Synthesis of Pentyl Acetate

In this work, the enzymatic synthesis of pentyl acetate obtained from acetic acid and pentan-1-ol using the commercial immobilized lipase Lipozyme®435 was studied. Specifically, the effects of several variables of the process on the kinetics were shown, such as the initial concentration of the acetic acid, the alcohol/acid molar ratio, and the possible reuse of the enzyme, while other variables, such as temperature, agitation, and the enzyme/acid ratio were held constant. The kinetics were determined by assessing the acetic acid concentration throughout the reactive process. Experimental data were correlated with the rate equation consisting of a modified version of the Bi–Bi Ping-Pong mechanism. The results showed that when no hydrophobic solvents were used with the reagents in stoichiometric proportion, a high molar fraction of acetic acid (x0,acid ≈ 0.50) caused the loss of enzymatic activity, achieving a conversion of only 5%. However, when there was an excess of pentan-1-ol, the reaction occurred successfully. Under optimal conditions (solvent-free conditions, x0,alcohol/x0,acid = 2, and x0,acid = 0.33), it was found that the enzyme could be reused up to 10 times without a loss of activity, reaching conversions higher than 80% after 8 h. Therefore, those conditions are advantageous in terms of productivity.