Structural Mimics Research Articles

A Structural and Functional Mimic of P680+

One or multiple chlorophyll molecules are employed in the reaction center of photosystem II's main electron donor (defined as P680). We have a poor understanding of how the reaction center facilitates water oxidation and the roles that mono‐ and/or multimeric chlorophyll groups play when P680 oxidizes a neighboring tyrosine in order to drive water oxidation at the oxygen evolving complex. We have prepared a dimeric MgII‐porphyrin complex [Mg2(BTPP)] (1, H4‐BTPP = 1,2‐bis‐(10,15,20‐triphenylporphyrin‐5‐yl)‐benzene) as a structural and functional mimic of the dimeric core of P680. 1 was oxidized by one‐electron to the corresponding π‐cation radical complex 2. The radical cation was characterized by UV‐Vis‐NIR, FT‐IR, and EPR spectroscopic techniques. 2 was shown to be reactive towards phenols to give the corresponding phenoxyl radicals, mimicking the reactivity of the P680 cation radical which oxidizes tyrosine to tyrosyl radical. Critically, the dimeric π‐cation radical showed markedly higher rates of proton coupled electron transfer oxidation (PCET) of phenols when compared to its monomeric counterpart [Mg(TPP)] (TPP = 5,10,15,20‐tetraphenylporphyrin). Our findings demonstrate that MgII‐porphyrin complexes are reliable mimics of photosynthetic PCET processes and suggest that photosynthetic reaction centers with multiple π‐conjugated complexes likely lower the barrier to PCET oxidation by π‐cation radical species.

A Structural and Functional Mimic of P680.

One or multiple chlorophyll molecules are employed in the reaction center of photosystem II's main electron donor (defined as P680). We have a poor understanding of how the reaction center facilitates water oxidation and the roles that mono- and/or multimeric chlorophyll groups play when P680 oxidizes a neighboring tyrosine in order to drive water oxidation at the oxygen evolving complex. We have prepared a dimeric MgII-porphyrin complex [Mg2(BTPP)] (1, H4-BTPP = 1,2-bis-(10,15,20-triphenylporphyrin-5-yl)-benzene) as a structural and functional mimic of the dimeric core of P680. 1 was oxidized by one-electron to the corresponding π-cation radical complex 2. The radical cation was characterized by UV-Vis-NIR, FT-IR, and EPR spectroscopic techniques. 2 was shown to be reactive towards phenols to give the corresponding phenoxyl radicals, mimicking the reactivity of the P680 cation radical which oxidizes tyrosine to tyrosyl radical. Critically, the dimeric π-cation radical showed markedly higher rates of proton coupled electron transfer oxidation (PCET) of phenols when compared to its monomeric counterpart [Mg(TPP)] (TPP = 5,10,15,20-tetraphenylporphyrin). Our findings demonstrate that MgII-porphyrin complexes are reliable mimics of photosynthetic PCET processes and suggest that photosynthetic reaction centers with multiple π-conjugated complexes likely lower the barrier to PCET oxidation by π-cation radical species.

New BDNF and NT-3 Cyclic Mimetics Concur with Copper to Activate Trophic Signaling Pathways as Potential Molecular Entities to Protect Old Brains from Neurodegeneration.

A low level of Neurotrophins (NTs), their Tyrosine Kinase Receptors (Trks), Vascular Endothelial Growth Factors (VEGFs) and their receptors, mainly VEGFR1 and VEGFR2, characterizes AD brains. The use of NTs and VEGFs as drugs presents different issues due to their low permeability of the blood-brain barrier, the poor pharmacokinetic profile, and the relevant side effects. To overcome these issues, different functional and structural NT mimics have been employed. Being aware that the N-terminus domain as the key domain of NTs for the binding selectivity and activation of Trks and the need to avoid or delay proteolysis, we herein report on the mimicking ability of two cyclic peptide encompassing the N-terminus of Brain Derived Growth Factor (BDNF), (c-[HSDPARRGELSV-]), cBDNF(1-12) and of Neurotrophin3 (NT3), (c-[YAEHKSHRGEYSV-]), cNT3(1-13). The two cyclic peptide features were characterized by a combined thermodynamic and spectroscopic approach (potentiometry, NMR, UV-vis and CD) that was extended to their copper(II) ion complexes. SH-SY5Y cell assays show that the Cu2+ present at the sub-micromolar level in the complete culture media affects the treatments with the two peptides. cBDNF(1-12) and cNT3(1-13) act as ionophores, induce neuronal differentiation and promote Trks and CREB phosphorylation in a copper dependent manner. Consistently, both peptide and Cu2+ stimulate BDNF and VEGF expression as well as VEGF release; cBDNF(1-12) and cNT3(1-13) induce the expression of Trks and VEGFRs.

Osteosarcoma growth on trabecular bone mimicking structures manufactured via laser direct write

 This paper describes the direct laser write of a photocurable acrylate-based PolyHIPE (High Internal Phase Emulsion) to produce scaffolds with both macro- and microporosity, and the use of these scaffolds in osteosarcoma-based 3D cell culture. The macroporosity was introduced via the application of stereolithography to produce a classical “woodpile” structure with struts having an approximate diameter of 200 μm and pores were typically around 500 μm in diameter. The PolyHIPE retained its microporosity after stereolithographic manufacture, with a range of pore sizes typically between 10 and 60 μm (with most pores between 20 and 30 μm). The resulting scaffolds were suitable substrates for further modification using acrylic acid plasma polymerisation. This scaffold was used as a structural mimic of the trabecular bone and in vitro determination of biocompatibility using cultured bone cells (MG63) demonstrated that cells were able to colonise all materials tested, with evidence that acrylic acid plasma polymerisation improved biocompatibility in the long term. The osteosarcoma cell culture on the 3D printed scaffold exhibits different growth behaviour than observed on tissue culture plastic or a flat disk of the porous material; tumour spheroids are observed on parts of the scaffolds. The growth of these spheroids indicates that the osteosarcoma behave more akin to in vivo in this 3D mimic of trabecular bone. It was concluded that PolyHIPEs represent versatile biomaterial systems with considerable potential for the manufacture of complex devices or scaffolds for regenerative medicine. In particular, the possibility to readily mimic the hierarchical structure of native tissue enables opportunities to build in vitro models closely resembling tumour tissue.

Razor clam (Pinna bicolor) structural mimics as drivers of epibenthic biodiversity; a manipulative experiment

Shellfish ecosystems facilitate important ecological functions and communities, but overexploitation and mismanagement have contributed to their decline worldwide. Within recent decades, coastal management efforts have increasingly sought to understand and reinstate valuable ecological functions provided by habitat-forming bivalves including oysters, mussels and pinnids. However, many bivalve species are critically understudied, limiting restoration and ecological engineering opportunities. Pinnids, specifically, are an underappreciated bivalve group, with razor clams (Pinna bicolor) forming dense aggregations, and potentially supporting important ecological functions. This study, conducted in an urban Australian estuary, was a manipulative experiment that investigated whether artificial razor clam shells could facilitate beneficial ecological functions through the provision of structural habitat. Specifically, we investigated the influence of intertidal benthic structures, including the micro-habitat influences of surface structure associated with mortality status (open or closed shell), and the short-term response of the benthic and epifaunal communities. Within 12 weeks, the structural razor clam mimics rapidly changed the aboveground ecological community, relative to the bare habitat controls. Both open and closed artificial shells provided a settlement surface for epiphytic organisms and supported enhanced epifaunal biodiversity. Contrastingly, the artificial structures did not significantly alter sediment characteristics or infaunal macroinvertebrate composition in the surrounding benthos. These results provide important insights into the rapid ecological response to the installation of intertidal pinnid structures in dynamic estuarine ecosystems. Furthermore, we provide a case study for understanding the ecological functions of an understudied habitat-forming species, which could be used to inform future restoration and management efforts.

Synthesis of Structural ADP-Ribose Analogues as Inhibitors for SARS-CoV-2 Macrodomain 1.

Protein adenosine diphosphate (ADP)-ribosylation is crucial for a proper immune response. Accordingly, viruses have evolved ADP-ribosyl hydrolases to remove these modifications, a prominent example being the SARS-CoV-2 NSP3 macrodomain, "Mac1". Consequently, inhibitors are developed by testing large libraries of small molecule candidates, with considerable success. However, a relatively underexplored angle in design pertains to the synthesis of structural substrate mimics. Here, we present the synthesis and biophysical activity of novel adenosine diphosphate ribose (ADPr) analogues as SARS-CoV-2 NSP3 Mac1 inhibitors.

Measuring Mass Transfer of n-Hexane and 2-Chloroethyl Ethyl Sulfide in Sorbent/Polymer Fiber Composites Using a Volumetric Adsorption Apparatus.

The integration of metal-organic frameworks (MOFs) into composite systems serves as an effective strategy to increase the processability of these materials. Notably, MOF/fiber composites have shown much promise as protective equipment for the capture and remediation of chemical warfare agents. However, the practical application of these composites requires an understanding of their mass transport properties, as both mass transfer resistance at the surface and diffusion within the materials can impact the efficacy of these materials. In this work, we synthesized composite fibers of MOF-808 and amidoxime-functionalized polymers of intrinsic microporosity (PIM-1-AX) and measured the adsorption and mass transport behavior of n-hexane and 2-chloroethyl ethyl sulfide (CEES), a sulfur mustard simulant. We developed a new Fickian diffusion model for cylindrical shapes to fit the dynamic adsorption data obtained from a commercial volumetric adsorption apparatus and found that mass transport behavior in composite fibers closely resembled that in the pure PIM fibers, regardless of MOF loading. Moreover, we found that n-hexane adsorption mirrors that of CEES, indicating that it could be used as a structural mimic for future adsorption studies of the sulfur mustard simulant. These preliminary insights and the new model introduced in this work lay the groundwork for the design of next-generation composite materials for practical applications.

Bioinformatics leading to conveniently accessible, helix enforcing, bicyclic ASX motif mimics (BAMMs).

Helix mimicry provides probes to perturb protein-protein interactions (PPIs). Helical conformations can be stabilized by joining side chains of non-terminal residues (stapling) or via capping fragments. Nature exclusively uses capping, but synthetic helical mimics are heavily biased towards stapling. This study comprises: (i) creation of a searchable database of unique helical N-caps (ASX motifs, a protein structural motif with two intramolecular hydrogen-bonds between aspartic acid/asparagine and following residues); (ii) testing trends observed in this database using linear peptides comprising only canonical L-amino acids; and, (iii) novel synthetic N-caps for helical interface mimicry. Here we show many natural ASX motifs comprise hydrophobic triangles, validate their effect in linear peptides, and further develop a biomimetic of them, Bicyclic ASX Motif Mimics (BAMMs). BAMMs are powerful helix inducing motifs. They are synthetically accessible, and potentially useful to a broad section of the community studying disruption of PPIs using secondary structure mimics.

Preparation and characterization of MnIIMnIII complexes with relevance to class Ib ribonucleotide reductases

The Mn2 complex [MnII2(TPDP)(O2CPh)2](BPh4) (1, TPDP = 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol, Ph =phenyl) was prepared and subsequently characterized via single-crystal X-ray diffraction, X-ray absorption, electronic absorption, and infrared spectroscopies, and mass spectrometry. 1 was prepared in order to explore its properties as a structural and functional mimic of class Ib ribonucleotide reductases (RNRs). 1 reacted with superoxide anion (O2•–) to generate a peroxido-MnIIMnIII complex, 2. The electronic absorption and electron paramagnetic resonance (EPR) spectra of 2 were similar to previously published peroxido-MnIIMnIII species. Furthermore, X-ray near edge absorption structure (XANES) studies indicated the conversion of a MnII2 core in 1 to a MnIIMnIII state in 2. Treatment of 2 with para-toluenesulfonic acid (p-TsOH) resulted in the conversion to a new MnIIMnIII species, 3, rather than causing O—O bond scission, as previously encountered. 3 was characterized using electronic absorption, EPR, and X-ray absorption spectroscopies. Unlike other reported peroxido-MnIIMnIII species, 3 was capable of oxidative O—H activation, mirroring the generation of tyrosyl radical in class Ib RNRs, however without accessing the MnIIIMnIV state.

A CoII-Hydroxide Complex That Converts Directly to a CoII-Acetamide during Catalytic Nitrile Hydration.

In exploring structural and functional mimics of nitrile hydratases, we report the synthesis of the pseudo-trigonal bipyramidal CoII complexes (K)[CoII(DMF)(LPh)] (1(DMF)), (NMe4)2[CoII(OAc)(LPh)] (1(OAc)), and (NMe4)2[CoII(OH)(LPh)] (1(OH)) (LPh = 2,2',2''-nitrilo-tris-(N-phenylacetamide; DMF = N,N-dimethylformamide; -OAc = acetate)). The complexes were characterized using NMR, FT-IR, ESI-MS, electronic absorption spectroscopy, and X-ray crystallography, showing the LPh ligand to bind in a tetradentate tripodal fashion alongside the respective ancillary donor. One of the complexes, 1(OH), is an unusual structural and functional mimic of the Co active site in Co nitrile hydratases. 1(OH) reacted with acetonitrile to yield the CoII-acetamide complex (NMe4)2[CoII(NHC(O)CH3)(LPh)], 2, which was also thoroughly characterized. In the presence of excess hydroxide, 1(OH) was found to catalyze quantitative conversion of the added hydroxide into acetamide. Despite the differences in Co oxidation state in nitrile hydratases and 1(OH) (CoIII versus CoII, respectively), 1(OH) was nonetheless an effective nitrile hydration catalyst, selectively producing acetamide over multiple turnovers.

Chemical Space Profiling of SARS-CoV-2 PLpro Using DNA-Encoded Focused Libraries.

DNA-encoded library (DEL) technology is gaining attention for its rapid construction and deconvolution capabilities. Our study explored a novel strategy using rational DELs tailored for the SARS-CoV-2 papain-like protease, which revealed new fragments. Structural changes post-DEL screening mimic traditional medicinal chemistry lead optimization. We unveiled unique aromatic structures offering an alternative optimization path. Notably, we identified superior binding fragments targeting the BL2 groove. Derivative 16 emerged as the most promising by exhibiting IC50 values of 0.25 μM. Derivative 6, which features an aromatic fragment capped with a naphthalene moiety, showed IC50 values of 2.91 μM. Molecular modeling revealed hydrogen bond interactions with Lys157 residue and potential covalent interactions with nearby amino acid residues. This research underscored DEL's potential for fragment-based drug discovery against SARS-CoV-2 protease.

How Do Differences in Electronic Structure Affect the Use of Vanadium Intermediates as Mimics in Nonheme Iron Hydroxylases?

We study active-site models of nonheme iron hydroxylases and their vanadium-based mimics using density functional theory to determine if vanadyl is a faithful structural mimic. We identify crucial structural and energetic differences between ferryl and vanadyl isomers owing to the differences in their ground electronic states, i.e., high spin (HS) for Fe and low spin (LS) for V. For the succinate cofactor bound to the ferryl intermediate, we predict facile interconversion between monodentate and bidentate coordination isomers for ferryl species but difficult rearrangement for vanadyl mimics. We study isomerization of the oxo intermediate between axial and equatorial positions and find the ferryl potential energy surface to be characterized by a large barrier of ca. 10 kcal/mol that is completely absent for the vanadyl mimic. This analysis reveals even starker contrasts between Fe and V in hydroxylases than those observed for this metal substitution in nonheme halogenases. Analysis of the relative bond strengths of coordinating carboxylate ligands for Fe and V reveals that all of the ligands show stronger binding to V than Fe owing to the LS ground state of V in contrast to the HS ground state of Fe, highlighting the limitations of vanadyl mimics of native nonheme iron hydroxylases.

Cyclization Scaffolding for Improved Vaccine Immunogen Stability: Application to Tau Protein in Alzheimer's Disease.

Effective scaffolding of immunogens is crucial for generating conformationally selective antibodies through active immunization, particularly in the treatment of protein misfolding diseases such as Alzheimer's and Parkinson's disease. Previous computational work has revealed that a disorder-prone region of the tau protein, when in a stacked form, is predicted to structurally resemble a small, soluble protofibril, having conformational properties similar to those of experimental in vitro tau oligomers. Such an oligomeric structural mimic has the potential to serve as a vaccine immunogen design for Alzheimer's disease. In this study, we developed a cyclization scaffolding method in Rosetta, in which multiple cyclic peptides are stacked into a protofibril. Cyclization results in significant stabilization of protofibril-like structures by constraining the conformational space. Applying this method to the disorder-prone region of the tau fibril, we evaluated the metastability of the cyclized tau immunogen using molecular dynamics simulations, and we identified sequences of two cyclic constructs having high metastability in the protofibril. We then assessed their thermodynamic stability by computing the free energy required to separate a distal chain from the rest of the stacked structure. Our computational results, based on molecular dynamics simulations and free energy calculations, demonstrate that two cyclized constructs, cyclo-(VKSEKLDFKDRVQSKIFyN) and cyclo-(VKSEKLDFKDRVQSKIYvG) (lowercase letters indicate d-form amino acids), possess significantly increased thermodynamic stability in the protofibril over an uncyclized linear construct VKSEKLDFKDRVQSKI. The cyclization scaffolding approach proposed here holds promise as a means to effectively design immunogens for protein misfolding diseases, particularly those involving liposome-conjugated peptide constructs.

Fluorescent carbazole-derived α-amino acids: structural mimics of tryptophan.

Fluorescent tags are commonly used for imaging of proteins and peptides during biological events; however, the large size of dyes can disrupt protein structure and function, and typically require the use of a chemical spacer. Herein, we report the synthesis of a new class of fluorescent unnatural α-amino acid, containing carbazole side-chains designed to mimic l-tryptophan and thus, readily incorporated into peptides. The amino acids were constructed using a Negishi cross-coupling reaction as the key step and exhibited strong fluorescent emission, with high quantum yields in both organic solvents and water. Compatible with solid phase peptide synthesis, the carbazole amino acids were used to replace tryptophan in a β-hairpin model peptide and shown to be a close structural mimic with retention of conformation. They were also found to be effective fluorescent molecular reporters for biological events. Incorporation into a proline-rich ligand of the WW domain protein demonstrated that the fluorescent properties of a carbazole amino acid could be used to measure the protein-protein binding interaction of this important biological signalling process.

Amphoteric reactivity of a putative Cu(II)-mCPBA intermediate.

In copper-based enzymes, Cu-hydroperoxo/alkylperoxo species are proposed as key intermediates for their biological activity. A vast amount of literature is available on the functional and structural mimics of enzymatic systems with heme and non-heme ligand frameworks to stabilize high valent metal intermediates, mostly at low temperatures. Herein, we report a reaction between [CuI(NCCH3)4]+ and meta-chloroperoxybenzoic acid (mCPBA) in CH3CN that produces a putative CuII(mCPBA) species (1). 1 was characterized by UV/Vis, resonance Raman, and EPR spectroscopies. 1 can catalyze both electrophilic and nucleophilic reactions, demonstrating its amphoteric behavior. Additionally, 1 can also conduct electron transfer reactions with a weak reducing agent such as diacetyl ferrocene, making it one of the reactive copper-based intermediates. One of the most important aspects of the current work is the easy synthesis of a CuII(mCPBA) adduct with no complicated ligands for stabilization. Over time, 1 decays to form a CuII paddle wheel complex (2) and is found to be unreactive towards substrate oxidation.

Synthetic hydrogel polymer nanoparticles as a structural mimic of carbonic anhydrase

Synthetic hydrogel polymer nanoparticles as a structural mimic of carbonic anhydrase

A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase

The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His)2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.

Rubredoxin Protein Scaffolds Sourced from Diverse Environmental Niches as an Artificial Hydrogenase Platform.

Nickel-substituted rubredoxin (NiRd) from Desulfovibrio desulfuricans has previously been shown to act as both a structural and functional mimic of the [NiFe] hydrogenase. However, improvements both in turnover frequency and overpotential are needed to rival the native [NiFe] hydrogenase enzymes. Characterization of a library of NiRd mutants with variations in the secondary coordination sphere suggested that protein dynamics played a substantial role in modulating activity. In this work, rubredoxin scaffolds were selected from diverse organisms to study the effects of distal sequence variation on catalytic activity. It was found that though electrochemical catalytic activity was only slightly impacted across the series, the Rd sequence from a psychrophilic organism exhibited substantially higher levels of solution-phase hydrogen production. Additionally, Eyring analyses suggest that catalytic activation properties relate to the growth temperature of the parent organism, implying that the general correlation between the parent organism environment and catalytic activity often seen in naturally occurring enzymes may also be observed in artificial enzymes. Selecting protein scaffolds from hosts that inhabit diverse environments, particularly low-temperature environments, represents an alternative approach for engineering artificial metalloenzymes.

Biomimetic Transition Metal Cluster Complexes: Synthetic Studies and Applications in the Reduction of Inert Small Molecules

Abstract Transition metal cluster complexes, particularly those containing iron and sulfur, are used as catalysts for the biological reduction of inert small molecules such as N2 and CO2. The structures of these biological clusters are complicated and the protein backbones around the clusters often play important roles in catalysis, hence reproducing or mimicking the enzymatic functions with synthetic cluster complexes remains a challenge. Appropriate assumptions and hypotheses on the relationships between the structures and functions of biological clusters are needed to develop synthetic molecular catalysts inspired by enzymes. This account reviews recent studies by the author and his coworkers on iron-containing biomimetic cluster complexes. Cubic Mo-Fe-S clusters supported by bulky cyclopentadienyl ligands on molybdenum were designed and synthesized, and their Fe sites captured and catalytically converted N2 under reducing conditions. Iron-hydride clusters, which are relevant to the active species of biological and industrial nitrogen fixation, also served as catalysts for the reduction of N2. Furthermore, various metal-sulfur clusters, ranging from a structural mimic of the complex active site of the N2-reducing enzyme to a simple and cubic [Fe4S4] cluster, were found to catalyze the biologically inaccessible direct conversion of CO2 to short-chain hydrocarbons. These studies have demonstrated the potential utility of biomimetic approaches to the catalytic reduction of inert small molecules, through the rational design and synthesis of simple yet appropriate iron-containing cluster complexes.

Biologically inspired 3Fe4S cluster as structural mimics of FeMoco M‐cluster

AbstractA 3Fe4S cluster related to M‐cluster of Mo‐nitrogenase is reported. [K(THF)5][Fe3(μ‐bdt)2(μ‐PPh2)(CO)5] (1) is synthesized from photo‐assisted structural rearrangement of [K(THF)2][(μ,κ2‐bdt)Fe2(μ‐PPh2)(CO)5] under visible light irradiation. The molecular structure of 1 consists of a Fe3 core bearing one dithiolate bridge and the second dithiolate group capping onto the metallic plane. The structural motif of 1 is analogous to that of Mo‐participated 3Fe4S unit in M‐cluster of Mo‐nitrogenase, with the similar Fe‐Fe and Fe‐S bond distances. Upon protonation in 193 K, a Fe‐hydride species (1H) is generated and characterized to possess the bridging hydride group (δ = −12.57 ppm) by 1H‐NMR spectroscopy and DFT calculation. In CH3CN solution, complex 1 exhibits a reversible reduction and oxidation process at = −1.94 V and = −0.18 V, respectively, at 273 K. The reduction behavior of 1 in CH2Cl2 solution at 243 K displays a slight modification in the presence of trifluoroacetic acid, revealing a moderate anodic potential shift (~50 mV). The current amplitude of the reduction wave is linearly increased with the increasement of acid added, indicative of the catalytic event.