Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy

Abstract

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as pnitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅ 1 ). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅ 2 ). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

Pre-Steady-State and Steady-State Kinetic Analysis of Butyrylcholinesterase-Catalyzed Hydrolysis of Mirabegron, an Arylacylamide Drug

“…This catalytic behavior may have important physiological, pharmacological and toxicological consequences, e.g., damping the response of targeted regulatory enzymes and enzymes located in micro-compartments where re-binding of the ligand/substrate may take place after dissociation of complexes. A recent work pointed out the importance of hysteretic (also called "allokairy") regulation of a promiscuous monomeric esterase [26]. Thus, the tight regulation of biological systems implies the fine tuning of enzyme activity for optimizing physiological responses.…”

3-Chymotrypsin-like Protease (3CLpro): Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials

“…Taken together, all results combinated with our bioinformatics data suggest that the catalytic mechanism of 3CLpro is formed by a His41-Cys145-Asp187 triad. Indeed, the proposal of the catalytic triad participating in reactions is classic in several catalytic reactions of various enzyme classes [64][65][66][67][68]. The proposed mechanism in this review follows two phases, acylation followed by deacylation, where the roles of these residues would be analogous to the catalytic triad of 3CLpro.…”

3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials

“…Taken together, all results combinated suggest that the catalytic mechanism of 3CLpro is formed by a His41-Cys145-Asp187 triad. Indeed, the proposal of the catalytic triad participating in reactions is classic in several catalytic reactions of various enzyme classes [ 61 , 62 , 63 , 64 , 65 ]. For example, QM-MM studies conducted by Arafet and co-authors showed that the residues Gln19, Asn175, and Trp181, located near His159 of the cruzain (the major protease of Trypanosoma cruzi ), are essential for modulating the p K a of the catalytic histidine, allowing it to act as acid and base in the catalysis [ 66 ].…”