Charge Relay System Research Articles

Charge Relay Without Proton Transfer: Coupling of Two Short Hydrogen Bonds via Imidazole in Models of Catalytic Triad of Serine Protease Active Site.

A homologous series of 20 substituted alcohol-imidazole-acetate model complexes imitating the charge relay system in Ser-His-Asp catalytic triad of serine proteases is considered quantum-chemically. We show qualitatively that the geometries of alcohol-imidazole and imidazole-acetate short hydrogen bonds are strongly coupled via the central imidazole and such complexes are capable of effectively relaying the charge from acetate to alcohol moiety upon relatively small concerted proton displacements. We hypothesize an alternative catalytic mechanism of serine proteases that does not require two complete proton transfers or hydrogen bond breakage between Ser and His residues.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. μs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Actions of Novel Angiotensin Receptor Blocking Drugs, Bisartans, Relevant for COVID-19 Therapy: Biased Agonism at Angiotensin Receptors and the Beneficial Effects of Neprilysin in the Renin Angiotensin System.

Angiotensin receptor blockers (ARBs) used in the treatment of hypertension and potentially in SARS-CoV-2 infection exhibit inverse agonist effects at angiotensin AR1 receptors, suggesting the receptor may have evolved to accommodate naturally occurring angiotensin ‘antipeptides’. Screening of the human genome has identified a peptide (EGVYVHPV) encoded by mRNA, complementary to that encoding ANG II itself, which is an inverse agonist. Thus, opposite strands of DNA encode peptides with opposite effects at AR1 receptors. Agonism and inverse agonism at AR1 receptors can be explained by a receptor ‘switching’ between an activated state invoking receptor dimerization/G protein coupling and an inverse agonist state mediated by an alternative/second messenger that is slow to reverse. Both receptor states appear to be driven by the formation of the ANG II charge-relay system involving TyrOH-His/imidazole-Carboxylate (analogous to serine proteases). In this system, tyrosinate species formed are essential for activating AT1 and AT2 receptors. ANGII is also known to bind to the zinc-coordinated metalloprotease angiotensin converting enzyme 2 (ACE2) used by the COVID-19 virus to enter cells. Here we report in silico results demonstrating the binding of a new class of anionic biphenyl-tetrazole sartans (‘Bisartans’) to the active site zinc atom of the endopeptidase Neprilysin (NEP) involved in regulating hypertension, by modulating humoral levels of beneficial vasoactive peptides in the RAS such as vasodilator angiotensin (1–7). In vivo and modeling evidence further suggest Bisartans can inhibit ANG II-induced pulmonary edema and may be useful in combatting SARS-CoV-2 infection by inhibiting ACE2-mediated viral entry to cells.

Discovery of a new generation of angiotensin receptor blocking drugs: Receptor mechanisms and in silico binding to enzymes relevant to SARS-CoV-2

The discovery and facile synthesis of a new class of sartan-like arterial antihypertensive drugs (angiotensin receptor blockers [ARBs]), subsequently referred to as “bisartans” is reported. In vivo results and complementary molecular modelling presented in this communication indicate bisartans may be beneficial for the treatment of not only heart disease, diabetes, renal dysfunction, and related illnesses, but possibly COVID-19. Bisartans are novel bis-alkylated imidazole sartan derivatives bearing dual symmetric anionic biphenyl tetrazole moieties. In silico docking and molecular dynamics studies revealed bisartans exhibited higher binding affinities for the ACE2/spike protein complex (PDB 6LZG) compared to all other known sartans. They also underwent stable docking to the Zn2+ domain of the ACE2 catalytic site as well as the critical interfacial region between ACE2 and the SARS-CoV-2 receptor binding domain. Additionally, semi-stable docking of bisartans at the arginine-rich furin-cleavage site of the SARS-CoV-2 spike protein (residues 681–686) required for virus entry into host cells, suggest bisartans may inhibit furin action thereby retarding viral entry into host cells. Bisartan tetrazole groups surpass nitrile, the pharmacophoric “warhead” of PF-07321332, in its ability to disrupt the cysteine charge relay system of 3CLpro. However, despite the apparent targeting of multifunctional sites, bisartans do not inhibit SARS-CoV-2 infection in bioassays as effectively as PF-07321332 (Paxlovid).

Receptor Interactions of Angiotensin II and Angiotensin Receptor Blockers-Relevance to COVID-19.

Angiotensin II (Ang II) may contain a charge relay system (CRS) involving Tyr/His/carboxylate, which creates a tyrosinate anion for receptor activation. Energy calculations were carried out to determine the preferred geometry for the CRS in the presence and absence of the Arg guanidino group occupying position 2 of Ang II. These findings suggest that Tyr is preferred over His for bearing the negative charge and that the CRS is stabilized by the guanidino group. Recent crystallography studies provided details of the binding of nonpeptide angiotensin receptor blockers (ARBs) to the Ang II type 1 (AT1) receptor, and these insights were applied to Ang II. A model of binding and receptor activation that explains the surmountable and insurmountable effects of Ang II analogues sarmesin and sarilesin, respectively, was developed and enabled the discovery of a new generation of ARBs called bisartans. Finally, we determined the ability of the bisartan BV6(TFA) to act as a potential ARB, demonstrating similar effects to candesartan, by reducing vasoconstriction of rabbit iliac arteries in response to cumulative doses of Ang II. Recent clinical studies have shown that Ang II receptor blockers have protective effects in hypertensive patients infected with SARS-CoV-2. Therefore, the usage of ARBS to block the AT1 receptor preventing the binding of toxic angiotensin implicated in the storm of cytokines in SARS-CoV-2 is a target treatment and opens new avenues for disease therapy.

Discovery of small-molecule enzyme activators by activity-based protein profiling.

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse – small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1 – a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical, and biophysical analyses indicate that this compound increases LYPLAL1’s catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

Simple Design of an Enzyme-Inspired Supported Catalyst Based on a Catalytic Triad

Summary Enzyme active sites afford an intricate interplay of functional groups to mediate complex organic and inorganic reactions. Many hydrolytic enzymes use a catalytic triad comprising three different functional residues—(Ser(-OH), Hist(-imidazole), Asp(-CO 2 H))—that catalyze the hydrolysis of numerous unique substrates. Inspired by this design, we have developed a simple one-step synthesis for preparing a new supported catalytic system in which the three reactive groups of the catalytic triad (alcohol, imidazole, and carboxylate) are incorporated into a single functional unit. These artificial active sites can be coupled to a solid-phase support (Merrifield resin) by copper(I)-catalyzed azide-alkyne cycloaddition "click chemistry," and their effectiveness as esterolysis catalysts was demonstrated. Furthermore, tuning the local hydrophobicity of the resin particles with an approach analogous to the native enzyme hydrophobic pocket increased the catalytic efficiency. Quantum mechanics and molecular dynamics computational modeling were used to probe the catalytic effect and suggested a concerted two-step mechanism and hydrophobic nanoenvironment similar to that of hydrolytic enzymes.

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.

Experimentally optimized threading structures of the proton-coupled folate transporter.

The proton‐coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X‐ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol‐3‐phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head‐groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward‐open. Several positions lining the substrate cavity have been identified. Motif A, a helix‐turn‐helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward‐open state. A charge‐relay system between three charged residues as well as apposing glycines in two α‐helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward‐open state of a putative proton‐driven transporter (YajR).

Modulating the cobalt redox potential through imidazole hydrogen bonding interactions in a supramolecular biomimetic protein-cofactor model.

A realistic model for the active site of histidine-on cobalamin@protein complexes is reported and studied under homogeneous and immobilized conditions. Analysis of lower ligand modulation and its influence on the properties of the biomimetic compound are presented. The cofactor attachment by a protein's histidine residue was imitated by covalently linking an artificial imidazole-containing linker to cobyric acid. The resulting intramolecular coordination complex is an excellent structural model of its natural archetype, according to 2D 1H-NMR studies and molecular modeling. The effect of deprotonation of the axially coordinating imidazole ligand - as proposed for natural cofactor complexes - tunes significantly the position of the cathodic peak (ΔV = -203 mV) and stabilizes thereby the CoIII form. Partial deprotonation of the imidazole moiety through hydrogen bonding interactions was then achieved by immobilizing the biomimetic model on hydrophobic C18 silica, which yielded an unprecedented insight on how this class of Cbl-dependent proteins may fine-tune their properties in biological systems.

In silico identification of 2-oxo-1,3-thiazolidine derivatives as novel inhibitor candidate of class II histone deacetylase (HDAC) in cervical cancer treatment

Cervical cancer ranks third among the most prevalent deadly cancer in women worldwide and ranks first in developing countries. It is caused by human papilloma virus (HPV) infection. Thus HDACs have become prominent inhibition target for cervical cancer treatment. In order to discover the new alternative HDAC inhibitors (HDACIs), we conducted a computer-aided drug discovery and development (CADDD) based on de novo approach. The compound library is based on 4-[(2-oxo-1,3-thiazolidin-3-yl)carbonyl] aniline. Screening of the best drug leads was evaluated from several parameters, such as docking and interaction analysis, pharmacology, in silico preclinical trial and molecular dynamics analysis. The inhibitory activity of these new designed ligands against Homo sapiens class II HDAC was determined by molecular docking simulation. Docking analysis yielded eight best ligands which have better binding affinity than the standards. Therefore, interaction analysis indicated that all best ligands performed coordination with Zn2+ cofactor in HDAC charge-relay system which are essential for HDAC inhibitory activities of these inhibitors. Pharmacology analysis and preclinical trials of these compounds including pharmacology properties, bioactivity, mutagenicity–carcinogenicity, absorption, distribution, metabolism, excretion and toxicity (ADMET) properties were done through in silico methods. Through this analysis, the best ligands meet Lipinski’s rule of five, have a better drug score than standards, and show good bioactivities, oral bioavailability and ADMET properties. All best ligands also have good synthetic accessibility and were proved to be new compounds that have never been synthesized before. Stability of HDAC–ligand complexes was also calculated through molecular dynamics (MD) simulation. Based on this simulation, complex of the best ligands with corresponding HDAC has a good stability based on RMSD (root mean square deviation) and interaction analysis. The study thus reveals eight best ligands (F, Ib14, O38, Kb17, Gd40, Aa50, Gc42 and Bb38) which have better binding affinity against human class II histone deacetylase (HDAC) through molecular docking, dynamics and interaction analysis. The best ligands were also found to have good bioactivities, oral bioavailability and ADMET properties through in silico pharmacology analysis and preclinical trial. These compounds were found to have a good synthetic accessibility; therefore they could be synthesized for further biological and clinical test.

208 Histone deacetylase (HDAC) I catalytic core Asp-His charge relay system is altered by HDAC inhibitors: atomistic simulation studies

208 Histone deacetylase (HDAC) I catalytic core Asp-His charge relay system is altered by HDAC inhibitors: atomistic simulation studies

Curcumin and hydroxamate-derivative (PCI-34058) interfere with histone deacetylase I catalytic core Asp-His charge relay system: atomistic simulation studies

Histone deacetylases (HDACs) are representative targets for the natural and synthetic chemicals used to transform cells to confer antitumor properties. In the current study, curcumin and hydroxamate-derivative PCI-34058-bound HDAC1 were subjected to atomistic simulation. The results support the view that fitting of curcumin and PCI-34058 within the HDAC1 pocket depends on extensive interactions between the aromatic moieties of the inhibitors and the extensive network of aromatic amino acid side chains lining the pocket of HDAC1. The interaction forces a local perturbation of the coiled structures connecting the pocket residues resulting in ligand-induced tightening of the pocket. In addition to the competitive occupancy of the histone-acetyl-lysine binding pocket by the inhibitors, interference with the in-pocket aspartate-histidine (ASP-HIS) charge relay system was also observed in inhibitor-bound HDAC1 systems. In conclusion, curcumin and PCI-34058-mediated ligand-dependent HDAC1 tunnel closure interferes negatively with the ASP-HIS charge relay system in HDAC1. Future design of HDAC inhibitors may benefit from optimizing competitive interaction with the ligand site and interference with the charge relay system.

Intrinsic flexibility of NLRP pyrin domains is a key factor in their conformational dynamics, fold stability, and dimerization.

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) are key proteins in the innate immune system. The 14 members of the NLRP family of NLRs contain an N-terminal pyrin domain which is central for complex formation and signal transduction. Recently, X-ray structures of NLRP14 revealed an unexpected rearrangement of the α5/6 stem-helix of the pyrin domain allowing a novel symmetric dimerization mode. We characterize the conformational transitions underlying NLRP oligomerization using molecular dynamics simulations. We describe conformational stability of native NLRP14 and mutants in their monomeric and dimeric states and compare them to NLRP4, a representative of a native pyrin domain fold. Thereby, we characterize the interplay of conformational dynamics, fold stability, and dimerization in NLRP pyrin domains. We show that intrinsic flexibility of NLRP pyrin domains is a key factor influencing their behavior in physiological conditions. Additionally, we provide further evidence for the crucial importance of a charge relay system within NLRPs that critically influences their conformational ensemble in solution.

Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.

Evidences for structural basis of altered ascorbate peroxidase activity in cadmium-stressed rice plants exposed to jasmonate

Binding interactions of cadmium (Cd) with rice ascorbate peroxidase (OsAPX) in presence or absence of jasmonate was examined in-silico. OsAPX is a 250 amino acid long protein with 90 % sequence similarity to soybean-APX. The 3D model of OsAPX obtained by homology modeling using soybean APX (PDBID:1OAF) as template was associated with -15975.85 kJ/mol energy, 100 % residues in favoured region, verify score of 0.85, ERRAT score 89.625 and a negative ProSA graph, suggesting OsAPX model to be of good quality, robust and reliable which was submitted with Protein Model Database with PMDBID: PM0078091. The rice ascorbate peroxidase ascorbate [OsAPX-Asc] complex had a substrate binding cavity involving residues at position (30)KSCAPL(35), (167)RCH(169) and (172)R wherein ascorbate accommodated via three H-bonds involving (30)Lys at the γ-edge of heme. (169)His served as a bridge between heme-porphyrin of OsAPX and ascorbate creating a charge relay system. Cd bound in [OsAPX-Asc-Cd] complex at (29)EKSCAPL(35), a site similar to ascorbate binding site. The binding of Cd caused breaking of (169)His bridge shifting the protein conformation. Cadmium exhibited four electrostatic interactions via (29)Glu of OsAPX backbone. Docking of [OsAPX-Asc] with jasmonic acid (JA) resulted in [OsAPX-Asc-JA] complex where 4-H-bonds held JA to OsAPX in a cavity at γ-edge on the distal side of heme. The binding of [OsAPX-Asc-JA] to Cd show the metal to bind at a position other than that involved in binding of OsAPX with Cd alone. Results indicate that Cd does not replace iron or ascorbate or JA but binds to OsAPX on the surface at a separate site electrostatically. In presence of JA the interactions involved in formation of [OsAPXAsc] are restored which is otherwise altered by the presence of Cd. The formation and reformation of H-bond take place between the [OsAPX-Asc] and Cd/JA. It is the interaction between heme and ascorbate which is modulated differently in presence of Cd/JA. In absence of JA, Cd-binds to the [OsAPX-Asc] complex at the proximal end of APX near Asc-binding site, whereas in presence of JA, Cd-binds on the opposite site of the Asc-binding site involving (30)Lys and (29)Glu residues. In-silico binding studies well correlate with the wet-lab results where exogenous application of JA increased the activity of OsAPX in rice grown under Cd-stress. Therefore it is concluded that the activity of OsAPX in rice roots and shoots are compromised under Cd-stress alone.

Disruption of Oligomerization and Dehydroalanine Formation as Mechanisms for ClpP Protease Inhibition

Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to their activity, such as the proteasome and the HIV protease. Here, we present a novel mechanism of protease inhibition that relies on active-site-directed small molecules that disassemble the protease complex. We show the applicability of this mechanism within the ClpP protease family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely inactive heptamers. Moreover, we report the selective β-sultam-induced dehydroalanine formation of the active site serine. This reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon β-sultam treatment. Collectively, these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active site or occupation of a regulatory allosteric site.

Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S(1) pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.

Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest

A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The Km-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK2, decreased with increasing [Ca2+]. The protease maintained considerable activity at the range of 10–60°C and pH 6.00–10.25, with optimum kcat/Km-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis–Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.

Substrate‐Induced Conformational Change and Isomerase Activity of Dienelactone Hydrolase and its Site‐Specific Mutants

Studies of the interactions of dienelactone hydrolase (DLH) and its mutants with both E and Z dienelactone substrates show that the enzyme exhibits two different conformational responses specific for hydrolysis of each of its substrate isomers. DLH facilitates hydrolysis of the Z dienelactone through an unusual charge-relay system that is initiated by interaction between the substrate carboxylate and an enzyme arginine residue that activates an otherwise non-nucleophilic cysteine. The E dienelactone does not display this substrate-arginine binding interaction, but instead induces an alternate conformational response that promotes hydrolysis. Furthermore, the substitution of cysteine 123 for serine (C123S) in DLH, instead of inactivating the enzyme as is typical for this active-site mutation, changes the catalysis from substrate hydrolysis to isomerisation. This is due to the deacylation of the acyl-enzyme intermediates being much slower, thereby increasing their lifetimes and allowing for their interconversion through isomerisation, followed by relactonisation.