Recognition–turnover coupling in transition-state-analogue-imprinted chymotrypsin-mimic polymers: unexplained control-polymer kinetics, active-site heterogeneity, and mixed template inhibition

A 2018 study by Mathew, Thomas and Devaky reported a molecularly imprinted polymer that mimics chymotrypsin: a highly crosslinked polymer carrying the serine-protease catalytic triad (histidine, serine, aspartic acid) was polymerized around a phosphonate transition-state analogue (TSA) template, and the resulting imprinted polymer (CM) hydrolysed chymotrypsin-specific dipeptides with a rate acceleration of about 1.7×10³ over uncatalysed hydrolysis and an imprinting efficiency of 45 over a non-imprinted control polymer (CP). The authors nevertheless acknowledged that they could not explain why the non-imprinted control sometimes showed a higher turnover number ($k_{cat}$) than the imprinted mimic, that the active sites are heterogeneous and their distribution and microscopic composition were uncharacterized, that the TSA template inhibited catalysis by a mixed rather than purely competitive mechanism whose thermodynamics were undetermined, and that catalytic proficiency remained orders of magnitude below the native enzyme. The most recent field reviews show that none of these questions has been resolved: a 2026 review of molecularly imprinted nanoreactors still lists binding-site heterogeneity and limited catalytic scope as persistent central design challenges. The open problem is to establish the mechanistic coupling between imprinting-defined molecular recognition and catalytic turnover in this class of artificial hydrolases.

Background

Molecular imprinting is a technique for creating synthetic recognition sites in polymer matrices: functional monomers are pre-organized around a template molecule, copolymerized with a large fraction of crosslinker, and the template is then removed, leaving cavities whose shape and arrangement of functional groups are complementary to the template. Molecularly imprinted polymers (MIPs) made this way are robust, cheap mimics of antibody- or enzyme-type binding.

To turn a binding MIP into a catalyst, the template is chosen as a transition-state analogue (TSA) — a stable molecule resembling the high-energy transition state of the target reaction — so that the imprinted cavity preferentially binds and stabilizes the transition state, the mechanism by which natural enzymes achieve rate enhancement. For hydrolytic enzymes this TSA is typically a phosphonate monoester, whose tetrahedral phosphonyl group mimics the tetrahedral intermediate of amide- or ester-bond hydrolysis. Chymotrypsin, the model enzyme in this problem, is a serine protease whose active site combines a catalytic triad of histidine, aspartate and serine residues (which relays protons to activate the serine as a nucleophile), a hydrophobic pocket that recognizes phenylalanine/tyrosine side chains, and an oxyanion hole that stabilizes the transition state; it accelerates peptide-bond hydrolysis by roughly $10^{10}$ over the uncatalysed reaction.

Catalysis is quantified by Michaelis–Menten parameters: the turnover number $k_{cat}$ (reactions per active site per unit time) and the specificity constant $k_{cat}/K_M$, where $K_M$ is the substrate concentration at half-maximal rate. For imprinted catalysts one additionally reports the rate acceleration $k_{acc}$ (catalysed over uncatalysed rate) and the imprinting efficiency $k_{im}$ (the ratio of the imprinted polymer’s rate acceleration to that of a non-imprinted control polymer prepared identically but without template, $k_{im}=k_{acc}(\mathrm{CM})/k_{acc}(\mathrm{CP})$). Inhibition mechanism is diagnosed from Lineweaver–Burk plots (double-reciprocal plots of rate versus substrate concentration): within an elementary single-site Michaelis–Menten scheme, purely competitive inhibition gives lines intersecting on the ordinate, while lines intersecting in the negative quadrant signal mixed inhibition, i.e. the inhibitor binds both the free catalyst and the catalyst–substrate complex with different affinities.

Mathew, Thomas and Devaky (2018) built an ethylene glycol dimethacrylate (EGDMA)-crosslinked chymotrypsin mimic: methacrylated amino-acid monomers supplied the His/Ser/Asp triad, imprinting with phenyl-1-benzyloxycarbonylaminoethylphosphonate created the TSA-complementary cavities, and the polymer (CM) hydrolysed dipeptides with C-terminal phenylalanine or tyrosine — the chymotrypsin-specific signature — with $k_{acc}\approx1.7\times10^{3}$ and $k_{im}\approx45$ relative to the non-imprinted control polymer CP. This sits within the historical range of catalytic MIPs: the strongest transition-state-imprinted systems, such as the carboxypeptidase-A-like polymer of Liu and Wulff combining an amidinium group with a zinc-ion binding site, reach accelerations of a few hundred fold, still orders of magnitude below natural enzymes, and a contemporary review (Nothling et al. 2019) noted that phosphonate-templated materials typically show modest enhancements. What distinguishes the 2018 study is that it exposes the internal puzzle of this field with unusual clarity: imprinting demonstrably creates recognition (45-fold imprinting efficiency, substrate discrimination), yet turnover does not reliably follow — the control polymer sometimes shows the larger $k_{cat}$ — and the authors stated they could not propose a clear explanation. Solving the mechanistic question requires confronting three interlocking features of the system that the authors themselves left open: which functional groups and site classes actually carry turnover; how the heterogeneous distribution and composition of active sites maps onto catalytic function; and what binding behaviour underlies the mixed rather than purely competitive inhibition pattern reported for the TSA template.

Problem Statement

For transition-state-analogue-imprinted chymotrypsin-mimic polymers of the type reported by Mathew, Thomas and Devaky (2018) — an EGDMA-crosslinked polymer bearing His/Ser/Asp catalytic functionality, imprinted with a phosphonate transition-state analogue — establish the mechanistic coupling between imprinting-defined molecular recognition and catalytic turnover. The observations that the coupling model must account for are those reported for the imprinted polymer CM and its non-imprinted control CP; the model is to be established for the composition class defined above — EGDMA-crosslinked, His/Ser/Asp-bearing, phosphonate-TSA-imprinted hydrolases — and is not to be restricted to the single reported polymer composition. Establishing the coupling requires resolving three interlocking questions that the 2018 study left open: (i) give a chemically specific explanation, at the level of functional groups and active-site classes, of how the reported imprinting efficiency of about 45 and chymotrypsin-specific substrate discrimination coexist with substrate measurements in which the non-imprinted control polymer CP exhibits a larger turnover number $k_{cat}$ than the imprinted mimic CM — reconciling the kinetic quantities and measurement conditions underlying the two reported figures, and identifying which catalytic entities (e.g. imidazole versus carboxyl versus hydroxyl groups, in imprinted versus random environments) carry the observed turnover; (ii) characterize the distribution and microscopic composition of the heterogeneous catalytic sites in the polymer and correlate them with catalytic function; and (iii) determine the binding thermodynamics and structural basis of the inhibition by the transition-state-analogue template, which the study diagnosed as mixed rather than purely competitive from Lineweaver–Burk lines intersecting in the negative quadrant, establishing what binding behaviour that pattern reflects in a heterogeneous multi-site polymer for which the elementary single-site interpretation is not adequate. The overarching objective is a mechanistic model of recognition–catalysis coupling in this mimic that accounts for all of these observations jointly and thereby explains why the catalytic proficiency of these polymers (about $10^{3}$ rate acceleration) remains orders of magnitude below that of native chymotrypsin (roughly $10^{10}$). The mechanistic model sought here must account jointly for (i)–(iii): it is to name the active-site classes and their relative contributions to turnover, reproduce both the reported imprinting efficiency ($k_{im}\approx45$) and the observation that the control polymer can exhibit the larger $k_{cat}$, and yield at least one testable prediction that distinguishes it from alternative mechanisms.

References

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