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Allostery, as seen in extant biology, governs the activity regulation of enzymes through the redistribution of conformational equilibria upon binding an effector. Herein, a minimal design is demonstrated where a dipeptide can exploit dynamic imine linkage to condense with simple aldehydes to access spherical aggregates as catalytically active states, which facilitates an orthogonal reaction due to the closer proximity of catalytic residues (imidazoles). The allosteric site (amine) of the minimal catalyst can concomitantly bind to an inhibitor via a dynamic exchange, which leads to the alternation of the energy landscape of the self-assembled state, resulting in downregulation of catalytic activity. Further, temporal control over allosteric regulation is realized via a feedback-controlled autonomous reaction network that utilizes the hydrolytic activity of the (in)active state as a function of time.

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In the early Earth, rudimentary enzymes must have utilized the available light energy source to modulate protometabolic processes. Herein, we report the light-responsive C-C bond manipulation via short peptide-based assemblies bound to the photosensitive molecular cofactor (azo-based photoswitch) where the energy of the light source regulated the binding sites which subsequently modulated the retro-aldolase activity. In the presence of a continual source of high-energy photons, temporal realization of a catalytically more proficient state could be achieved under nonequilibrium conditions. Further, the hydrophobic surface of peptide assemblies facilitated the binding of an orthogonal molecular catalyst that showed augmented activity (promiscuous hydrolytic activity) upon binding. This latent activity was utilized for the in situ generation of light-sensitive cofactor that subsequently modulated the retro-aldolase activity, thus creating a reaction network.

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Peptide-based biomimetic catalysts are promising materials for efficient catalytic activity in various biochemical transformations. However, their lack of operational stability and fragile nature in non-aqueous media limit their practical applications. In this study, we have developed a cladding technique to stabilize biomimetic catalysts within porous covalent organic framework (COF) scaffolds. This methodology allows for the homogeneous distribution of peptide nanotubes inside the COF (TpAzo and TpDPP) backbone, creating strong noncovalent interactions that prevent leaching. We synthesized two different peptide-amphiphiles, C10FFVK and C10FFVR, with lysine (K) and arginine (R) at the C-termini, respectively, which formed nanotubular morphologies. The C10FFVK peptide-amphiphile nanotubes exhibit enzyme-like behavior and efficiently catalyze C-C bond cleavage in a buffer medium (pH 7.5). We produced nanotubular structures of TpAzo-C10FFVK and TpDPP-C10FFVK through COF cladding by using interfacial crystallization (IC). The peptide nanotubes encased in the COF catalyze C-C bond cleavage in a buffer medium as well as in different organic solvents (such as acetonitrile, acetone, and dichloromethane). The TpAzo-C10FFVK catalyst, being heterogeneous, is easily recoverable, enabling the reaction to be performed for multiple cycles. Additionally, the synthesis of TpAzo-C10FFVK thin films facilitates catalysis in flow. As control, we synthesized another peptide-amphiphile, C10FFVR, which also forms tubular assemblies. By depositing TpAzo COF crystallites on C10FFVR nanotubes through IC, we produced TpAzo-C10FFVR nanotubular structures that expectedly did not show catalysis, suggesting the critical role of the lysines in the TpAzo-C10FFVK.

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Extant proteins exploit thermodynamically activated negatively charged coenzymes and hydrotropes to temporally access mechanistically important conformations that regulate vital biological functions, from metabolic reactions to expression modulation. Herein, we show that a short amyloid peptide can bind to a small molecular coenzyme by exploiting reversible covalent linkage to polymerize and access catalytically proficient nonequilibrium amyloid microphases. Subsequent hydrolysis of the activated coenzyme leads to depolymerization, realizing a variance of the surface charge of the assembly as a function of time. Such temporal change of surface charge dynamically modulates catalytic activities of the transient assemblies as observed in highly evolved modern-day biocatalysts.

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During the billions of years of the evolutionary journey, primitive polymers, involved in proto metabolic pathways with low catalytic activity, played critical roles in the emergence of modern enzymes with remarkable substrate specificity. The precise positioning of amino acid residues and the complex orchestrated interplay in the binding pockets of evolved enzymes promote covalent and non-covalent interactions to foster a diverse set of complex catalytic transformations. Recent efforts to emulate the structural and functional information of extant enzymes by minimal peptide based assemblies have attempted to provide a holistic approach that could help in discerning the prebiotic origins of catalytically active binding pockets of advanced proteins. In addition to the impressive sets of advanced biochemical transformations, catalytic promiscuity and cascade catalysis by such small molecule based dynamic systems can foreshadow the ancestral catalytic processes required for the onset of protometabolism. Looking beyond minimal systems that work close to equilibrium, catalytic systems and compartments under non-equilibrium conditions utilizing simple prebiotically relevant precursors have attempted to shed light on how bioenergetics played an essential role in chemical emergence of complex behaviour. Herein, we map out these recent works and progress where diverse sets of complex enzymatic transformations were demonstrated by utilizing minimal peptide based self-assembled systems. Further, we have attempted to cover the examples of peptide assemblies that could feature promiscuous activity and promote complex multistep cascade reaction networks. The review also covers a few recent examples of minimal transient catalytic assemblies under non-equilibrium conditions. This review attempts to provide a broad perspective for potentially programming functionality via rational selection of amino acid sequences leading towards minimal catalytic systems that resemble the traits of contemporary enzymes.

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Herein, we report the substrate induced generation of a transient catalytic microenvironment from a single amino acid functionalized fatty acid in presence of a cofactor hemin. The catalytic state accessed under non-equilibrium conditions showed acceleration of peroxidase activity resulting in degradation of the substrate and subsequently led to disassembly. Equilibrated systems could not access the three-dimensional microphases and showed substantially lower catalytic activity. Further, the assembled state showed latent catalytic function (promiscuity) to hydrolyze a precursor to yield the same substrate. Consequently, the assembly demonstrated protometabolism by exploiting the peroxidase-hydrolase cascade to augment the lifetime and the mechanical properties of the catalytic state.

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Early evolution benefited from a complex network of reactions involving multiple C-C bond forming and breaking events that were critical for primitive metabolism. Nature gradually chose highly evolved and complex enzymes such as lyases to efficiently facilitate C-C bond formation and cleavage with remarkable substrate selectivity. Reported here is a lipidated short peptide which accesses a homogenous nanotubular morphology to efficiently catalyze C-C bond cleavage and formation. This system shows morphology-dependent catalytic rates, suggesting the formation of a binding pocket and registered enhancements in the presence of the hydrogen-bond donor tyrosine, which is exploited by extant aldolases. These assemblies showed excellent substrate selectivity and templated the formation of a specific adduct from a pool of possible adducts. The ability to catalyze metabolically relevant cascade transformations suggests the importance of such systems in early evolution.

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Herein, we report the generation of simple condensates of short peptides with ATP, which are spatiotemporally formed under dissipative conditions created in presence of ATP-ase. These coacervates could imbibe cytochrome c and temporally modulate a redox reaction catalyzed by the entrapped protein, thus mimicking the advanced functional machinery of transient intercellular membraneless condensates of large proteins and RNA.

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