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

Abstract

•Hydrolase-inspired catalyst based on a ubiquitous active site: the catalytic triad•A versatile synthesis enables immobilization of catalyst onto engineered supports•Inspired by enzyme hydrophobic pockets, hydrophobic modification increases catalysis•Computational modeling supports a concerted mechanism similar to that of native enzymes The remarkable catalytic performance of enzymes has enabled their use in many industrial applications. However, the inherent instability and restricted operating window of enzymes limit their application. The development of more robust catalysts inspired by enzymes is a significant step toward the United Nations Sustainable Developmental Goals for responsible consumption and production. The catalytic triad is a textbook example of the complex chemistry that enzymes control at their active sites. We report an approach to enzyme-inspired catalysis by combining the three groups of the catalytic triad into a single molecule through a versatile, one-step procedure. We show that by coupling these active sites onto hydrophobically modified polymer resins, we can tune the active site’s local environment to increase catalysis. In addition to new industrial catalysts, this study provides insight into the mechanism of enzymes, highlighting the concerted action of multiple functional groups to afford catalysis. 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(-CO2H))—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. 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(-CO2H))—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. Nature has evolved efficient and highly orthogonal chemical strategies to control complex multi-step chemical processes. This control has been optimized for many different chemical transformations, and the specificity of enzymatic reactions is critical to many biological processes. In contrast, the design and performance of synthetic catalysts that mimic enzymes in both range and efficiency of the chemical transformations continues to be a major challenge. To address this challenge, we drew our attention to esterases, which are important industrial and academic enzymes with broad applicability from food and paper manufacturing to detergent formulations and pharmaceuticals.1Hasan F. Shah A.A. Hameed A. Industrial applications of microbial lipases.Enzyme Microb. Technol. 2006; 39: 235-251Crossref Scopus (1517) Google Scholar The mechanism of many esterases is similar to the classic serine protease example of chymotrypsin,2Hedstrom L. Serine protease mechanism and specificity.Chem. Rev. 2002; 102: 4501-4524Crossref PubMed Scopus (1343) Google Scholar whereby a catalytic triad at the active site modulates the reaction. This textbook example of an enzyme active site consists of serine, histidine, and aspartate residues and has been studied extensively with the aspartate forming a hydrogen bond with the imidazole ring of the histidine, which in turn deprotonates the alcohol of the serine to form a potent nucleophile.3Polgár L. The catalytic triad of serine peptidases.Cell. Mol. Life Sci. 2005; 62: 2162-2172Crossref Scopus (312) Google Scholar A key enzymatic feature that is clearly demonstrated in these triad systems is the presence of a ternary structure, which both generates a hydrophobic substrate-binding pocket and optimally positions the triad residues in close proximity. Building on this bio-inspiration, a long-standing opportunity in catalyst design has been to develop simple synthetic systems that can perform similar chemistry without the need for well-defined tertiary structures. Pioneering work by Breslow and Overman,4Breslow R. Overman L.E. ‘Artificial enzyme’ combining a metal catalytic group and a hydrophobic binding cavity.J. Am. Chem. Soc. 1970; 92: 1075-1077Crossref PubMed Scopus (310) Google Scholar Breslow,5Breslow R. Biomimetic chemistry and artificial enzymes - catalysis by design.Acc. Chem. Res. 1995; 28: 146-153Crossref Scopus (887) Google Scholar and Overberger et al.6Overberger C.G. Salamione J.C. Yaroslavsky S. 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(Camb.). 2012; 48: 11695-11697Crossref PubMed Scopus (31) Google Scholar In designing enzyme-inspired catalysts (EICs) based on esterases, our attention was drawn to the challenge of introducing all three functional groups of the catalytic triad—carboxylate, imidazole, and hydroxyl groups—into a single compound. Importantly, we present a simple and versatile design strategy for the preparation of a supported EIC with esterolytic activity from readily available starting materials. The relative arrangement of functional groups can be further tuned via judicious selection of the starting material. In addition to the functional units of the catalytic triad, an acetylene group is also incorporated into the overall design for orthogonal attachment to various solid supports. To illustrate the power and versatility of this approach, we designed, synthesized, and evaluated the esterolytic activty of a range of functionalized Merrifield resins26Merrifield R.B. Solid phase peptide synthesis, I. The synthesis of a tetrapeptide.J. Am. Chem. Soc. 1963; 85: 2149-2154Crossref Scopus (6699) Google Scholar that mimic the chemistry of the catalytic triad (Figure 1). We also developed and evaluated control structures, where the three groups of the catalytic triad were systematically eliminated, to confirm the importance of all three functional moieties (Figure 2D). We used computational modeling of the reaction to support both the formation of a hydrophobic local environment and the sequence of a two-step, acylation-deacylation catalytic mechanism, similar to that performed by hydrolytic enzymes.Figure 2Synthesis and Immobilization of an Artificial Catalytic TriadShow full caption(A) One-step synthesis of the protected artificial triad (M1) with a simple ring opening of a functional epoxide with a protected histidine analog.(B) Immobilization of the artificial triad onto modified Merrifield resin by copper-catalyzed azide acetylene click chemistry and subsequent deprotection to yield catalyst resins R1–R6.(C) Inclusion of a C16 alkyl group to tune the resin’s local hydrophobicity with a second click reaction and subsequent deprotection to yield hydrophobically modified resins R7–R11 (refer to Figures S1 and S2).(D) Alternative mono-, di-, and tri-functionalized catalytic control structures used for confirming the concerted performance of the triad design during catalysis (protecting groups: trt, trityl (triphenylmethyl); Boc, tert-butyl).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) One-step synthesis of the protected artificial triad (M1) with a simple ring opening of a functional epoxide with a protected histidine analog. (B) Immobilization of the artificial triad onto modified Merrifield resin by copper-catalyzed azide acetylene click chemistry and subsequent deprotection to yield catalyst resins R1–R6. (C) Inclusion of a C16 alkyl group to tune the resin’s local hydrophobicity with a second click reaction and subsequent deprotection to yield hydrophobically modified resins R7–R11 (refer to Figures S1 and S2). (D) Alternative mono-, di-, and tri-functionalized catalytic control structures used for confirming the concerted performance of the triad design during catalysis (protecting groups: trt, trityl (triphenylmethyl); Boc, tert-butyl). Key to this work is the facile synthesis of multi-functional building blocks as the starting motif. The utilization of a protected histidine derivative (H-Hist(1-Trt)-OtBu) conveniently affords access to two of the functional groups of the catalytic triad: the imidazole and carboxylate groups. The hydroxyl group is then introduced by simple ring opening of an acetylene-functionalized epoxide (1) by the primary amine of the protected histidine (2) (Figure 2A). Versatility is afforded here by the wide range of available starting materials, giving access to a myriad of unique EIC dyad and triad arrangements (Figure 2D). A single synthetic step leads to a multi-functional building block containing the three groups of the catalytic triad together with an acetylene group for orthogonal conjugation to a wide variety of surfaces via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry. Initially, commercial Merrifield resin (3.0 mmol g−1 loading, polystyrene cross-linked with 1% divinylbenzene, 100–200 mesh)27Merrifield R.B. Solid-phase peptide synthesis.Adv. Enzymol. Relat. Areas Mol. Biol. 1969; 32: 221-296PubMed Google Scholar was chosen as the support. Quantitative reaction with sodium azide then afforded the desired azide functional resin particles (3.0 mmol g−1 of azide), which could be coupled with the triad functional molecule (M1) under orthogonal CuAAC click chemistry conditions. This afforded access to a range of resins with different loading of the catalytic unit (Figure 1). The combination of monitoring the attenuation of the azide group (2,100 cm−1) by Fourier transform infrared measurements and secondary coupling with an acetylene-functionalized coumarin dye followed by UV-visible (UV-vis) spectroscoopy analysis, allows the level of M1 incorporation into the resin support to be quantified and confirmed (Figures S1 and S2). Treatment of the functional resins with trifluoroacetic acid (TFA) then removes the tert-butyl and trityl protecting groups to yield the free acid, alcohol, and imidazole functional groups immobilized on the polystyrene resin support. The versatile nature of this process allows a range of particles with M1 loading varying from 0.1 to 3 mmol g−1 to be prepared (R1–R6). We could then screen esterase activity by observing the hydrolysis of p-nitrophenyl butyrate (PNB) in the presence of the enzyme-inspired catalytic resins (R1–R6). In contrast to the traditional p-nitrophenyl acetate model system, PNB is significantly more stable under the assay conditions and has negligible background hydrolysis, making PNB a more challenging and realistic target. Significantly, esterase-like catalysis was observed for all EICs (R1–R6); however, it was not observed for the catalytic control structures lacking an element of the catalytic triad (Figure 3A). The stepwise nature of the resin functionalization procedure afforded access to a wide series of candidate materials, which were then evaluated for esterolysis. Resins containing the artificial triad functionality—with and without hydrophobic modification—as well as control structures lacking an element of the artificial triad, were all investigated. The specific triad arrangement (M1) was selected for further analysis given that the alternate triad and dyad candidates exhibited little esterolytic effect. We then implemented the well-established Michaelis-Menten model for enzyme kinetics to compare the reaction kinetics for each of the catalytic materials (Table 1). The first target was to vary the degree of artificial catalytic triad functionalization on the resin support (0.6–3.0 mmol g−1) in order to understand the role of increasing artificial active sites for catalysis. Figure 3B shows the initial velocity (first 100 s) for the esterolysis of PNB with resins with varying levels of incorporation of the artificial catalytic triad molecule only. Interestingly, a bell-shaped curve was obtained with the fastest reaction rates for intermediate loading values of 1.5 mmol g−1 (R3), which represents ∼50% functionalization of the available azides with the triad molecule (M1). This was further supported by the finding that the material exhibited the largest kcat value for substrate turnover (5.4 hr−1) of all the triad-only resins. Furthermore, the resin with 0.6 mmol g−1 triad loading (R1) was a more effective catalyst than the resin with a loading of 3 mmol g−1 (R6) in terms of both turnover (kcat = 3.4 versus 0.07 hr−1) and catalyst efficiency (kcat/KM 4.05 versus 0.09; valid because of the similar KM parameters between materials).28Eisenthal R. Danson M.J. Hough D.W. Catalytic efficiency and kcat/KM: a useful comparator?.Trends Biotechnol. 2007; 25: 247-249Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar This suggests that simple catalyst concentration does not dictate the catalytic efficiency of these enzyme mimics. An analogy can be drawn with the active site of the enzymes, where many factors, such as the hydrophobic nanoenvironment around the active site, contribute to performance. In these initial examples, increased loading of the triad unit significantly increases the hydrophilicity of the resin, which could in turn interrupt the affinity of this material for the hydrophobic substrate and decrease catalytic efficiency.Table 1Michaelis-Menten Rate Parameters of Resins with Different Functional GroupsCatalystCatalytic Triad Loading (mol g−1)Hexadecyne Loading (mol g−1)kcat (hr−1)KM (mM)kcat/KM (min−1 M−1)R10.6–3.4144.05R20.9–2.5341.23R31.5–5.4452.00R42.1–1.9171.86R52.7–0.10150.11R63.0–0.07130.09R72.40.65.8402.41R82.10.99812136.1R91.51.58.37304.65R100.92.10.52530.16R110.62.40.40100.67 Open table in a new tab For examination of this feature, the modularity of our synthetic approach allowed the polarity of the supported resins to be tuned by conjugation of C16 alkyl chains onto the resin, which effectively regulated the hydrophobicity of the catalytic local environment. The same series of resins was used as described above, with one modification; the unreacted azide groups of the resin (R1–R6) were reacted in a second step with hexadecyne under CuAAC conditions (R7–R11) (Figure 2C). Introducing C16 alkyl chains into the resin particles allowed fine control of their hydrophobicity and tuning of the catalytic environment (Figure 1). These hydrophobically modified EIC materials were then screened for esterolytic catalysis with PNB; the velocity during the burst phase of these hydrolysis reactions is displayed in Figure 3B. Significantly, introduction of the unreactive C16-alkyl chain led to consistently increased rates for the esterolysis reaction of PNB. For the functionalization levels studied here, optimized conditions were observed for a 2.1 mmol g−1 loading of M1 and a 0.9 mmol g−1 loading of hexadecyne (R8). This optimized material exhibited a catalytic rate enhancement in excess of 50 times that of the same resin without hydrophobic tuning (R4) (kcat 98 versus 1.9 hr−1). Figure 3C further highlights the impact of hydrophobic tuning; the catalytic sites of resin R8 display enhanced substrate turnover as a result of the inclusion of the hexadecyl group. Once again, a bell-shaped curve was observed for rate enhancements of the hydrophobically modified resins, indicating that a trade-off between catalytic and hydrophobic groups is required for optimized catalysis. These results suggest that hydrophobicity plays an important role in the efficiency of the synthetic triad catalyst. The exact cause is under investigation, but it is postulated that varying the hydrophobicity of the resin might result in increased affinity for the hydrophobic substrate and regulate the local pKa of the charged triad groups, leading to an increase in catalyst performance. The importance of all three functional groups for catalytic function was further investigated synthetically. We applied chemical design to systematically remove the three functional groups of the EIC to investigate their individual effects on catalysis experimentally. Figure 2D shows some of the structures investigated. Significantly, in all examples, including the fully protected (P1, which includes only N(α)H), mono-functional (M1, which only includes the O(γ) group), and di-functional (D1 and D2) resins, catalysis was essentially at background levels. Figure 3C further highlights the negligible esterolytic enhancement of the control structure D2, which has a dyad-like structure including an imidazole and carboxylate but lacking the hydroxyl group. These control structures, paired with the catalytic enhancements of the hydrophobic resins, indicate that both inclusion of all three groups of the EIC and tuning the local nanoenvironment play important roles in catalysis. To further probe the functional group interactions responsible for catalysis, we were motivated to conduct a thorough examination of the mechanism by which the resin-supported EIC catalyzes the hydrolysis of PNB substrate through quantum mechanics (QM) calculations (refer to Supplemental Experimental Procedures, Document S1. Supplemental Experimental Procedures, Figures S1–S16, and Tables S1–S5, Table S6. QM Coordinates for the Optimized Structures of Styrene-Free Species along the N(α)H Pathway, Related to Figures 4 and 6, Table S7. QM Coordinates for the Optimized Structures of Styrene-Free Species along the Imidazole Pathway, Related to Figures 4 and 6, Table S8. QM Coordinates for the Optimized Structures of Styrene-Free Species along the Uncatalyzed Pathway, Related to Figures 4 and 6, Table S9. QM Coordinates for the Lowest-Energy Structures of Styrene-Attached EIC and Substrate in the Gas Phase, Related to Figures 4 and 6, Table S10. QM Coordinates for the Optimized Structures of Styrene-Attached Species along the N(α)H Pathway, Related to Figures 4 and 6, Table S11. QM Coordinates for the Optimized Structures of Styrene-Supported Species along the Imidazole Pathway, Related to Figures 4 and 6, and Figures S5–S16). Because the synthetic design of the catalyst was inspired by the native Ser-His-Asp active-site triad found in a number of enzymes, such as the serine peptidase family,3Polgár L. The catalytic triad of serine peptidases.Cell. Mol. Life Sci. 2005; 62: 2162-2172Crossref Scopus (312) Google Scholar, 29Paetzel M. Dalbey R.E. Catalytic hydroxyl/amine dyads within serine proteases.Trends Biochem. Sci. 1997; 22: 28-31Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 30Topf M. Várnai P. Richards W.G. Ab initio QM/MM dynamics simulation of the tetrahedral intermediate of serine proteases: insights into the active site hydrogen-bonding network.J. Am. Chem. Soc. 2002; 124: 14780-14788Crossref PubMed Scopus (81) Google Scholar we initially examined a two-step mechanistic pathway analogous to that exhibited by these enzymes, which involves successive acylation and deacylation steps (Figure 4, top). We discovered, however, that to proceed via this pathway, the catalyst would have to adapt a very strained configuration to facilitate the necessary hydroxyl-imidazole-carboxyl group interactions that mimic the Ser-His-Asp interactions in enzymes. This is seen in Figure 5A, which shows the so-called near-attack conformer (NAC), which is the conformation of the substrate-enzyme complex that is poised for the subsequent reactions, in this case, initial base catalysis by imidazole and nucleophilic attack by O(γ) in a concerted manner similar to that of enzymes. The unfavorability of this NAC highlights the important role of the enzyme in both binding the reacting groups in close proximity and orientating them correctly for reaction; designing synthetic analogs that can achieve this poses an additional challenge, and further work is underway in this direction.Figure 5EIC Conformation Affording Catalysis and Hydrophobic InteractionsShow full caption(A and B) Near-attack conformation of (A) the N(α) pathway and (B) the imidazole pathway. These are the conformations of the substrate-EIC complexes that are poised for reaction. In the NAC for the imidazole pathway (relative conformer energy = 0 kcal mol−1), the O(γ)H group of the EIC forms a strong intra-molecular hydrogen bond (H bond) with N(δ) of the imidazole ring, and the carbonyl carbon (of the substrate) is only 2.69 Å away from O(γ), thus making it convenient for the initial base catalysis by imidazole and nucleophilic attack by O(γ). In the NAC of the N(α)-mediated scheme (relative conformer energy = −18 kcal mol−1), the O(γ)H group forms a H bond with N(α) of the catalyst (which then acts as the base), and the carbonyl carbon of the substrate is still conveniently located (within 3 Å) in relation to O(γ). These structures were identified from a full conformational search carried out for the smaller catalyst-substrate model without polymer support (refer to Figures S5–S8). The color representations are as follows: carbon, gray; oxygen, red; nitrogen, blue; and hydrogen, white.(C) Representative conformations for the hydrophobically modified resins with zero, two, and four styrene units separating the EIC and alkyl chain residues (EIC-0, EIC-2, and EIC-4, respectively). The C16 chain (green) can be seen to wrap closely around the EIC unit (violet) irrespectively of residue spacing, highlighting the formation of a hydrophobic pocket around the EIC groups. The most populated cluster C1 for each fragment is represented here (refer to Figures S3 and S4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Near-attack conformation of (A) the N(α) pathway and (B) the imidazole pathway. These are the conformations of the substrate-EIC complexes that are poised for reaction. In the NAC for the imidazole pathway (relative conformer energy = 0 kcal mol−1), the O(γ)H group of the EIC forms a strong intra-molecular hydrogen bond (H bond) with N(δ) of the imidazole ring, and the carbonyl carbon (of the substrate) is only 2.69 Å away from O(γ), thus making it convenient for the initial base catalysis by imidazole and nucleophilic attack by O(γ). In the NAC of the N(α)-mediated scheme (relative conformer energy = −18 kcal mol−1), the O(γ)H group forms a H bond with N(α) of the catalyst (which then acts as the base), and the carbonyl carbon of the substrate is still conveniently located (within 3 Å) in relation to O(γ). These structures were identified from a full conformational search carried out for the smaller catalyst-substrate model without polymer support (refer to Figures S5–S8). The color representations are as follows: carbon, gray; oxygen, red; nitrogen, blue; and hydrogen, white. (C) Representative conformations for the hydrophobically modified resins with zero, two, and four styrene units separating the EIC and alkyl chain residues (EIC-0, EIC-2, and EIC-4, respectively). The C16 chain (green) can be seen to wrap closely around the EIC unit (violet) irrespectively of residue spacing, highlighting the formation of a hydrophobic pocket around the EIC groups. The most populated cluster C1 for each fragment is represented here (refer to Figures S3 and S4). Nonetheless, although the NAC for the imidazole catalyzed process is very strained, we discerned an alternative conformer that lies some 18 kcal mol−1 lower in energy. In this case, the O(γ)H group of the EIC forms a H bond with the secondary amine N(α) of the catalyst rather than the N(δ) of the imidazole ring (Figure 5B). The close proximity of the O(γ)H and N(α) groups on the EIC residue helps to drive the formation of a H bond between these groups over the more distant and sterically strained imidazole nitrogen N(δ). This NAC leads to an alternate reaction pathway (Figure 4, bottom) in which the secondary amine N(α) of EIC acts as the specific base catalyst in place of the imidazole ring, reminiscent of the Lys-Ser catalytic dyad seen in a range of serine protease enzymes.31Feldman A.R. Lee J. Delmas B. Paetzel M. Crystal structure of a novel viral protease with a serine/lysine catalytic dyad mechanism.J. Mol. Biol. 2006; 358: 1378-1389Crossref PubMed Scopus (58) Google Scholar, 32Ekici O.D. Paetzel M. Dalbey R.E. Unconventional serine/threonine proteases: variations on the catalytic Ser/His/Asp triad configuration.Protein Sci. 2008; 17: 2023-2037Crossref PubMed Scopus (218) Google Scholar To distinguish these two pathways, we refer to the first as being imidazole mediated and the second as being N(α) mediated. We carried out theoretical calculations on all of the model structures (Figure 4) to determine the energy barriers along the different reaction pathways; we also studied the corresponding uncatalyzed reaction under the same conditions for comparison. We performed calculations in the gas phase because it is most representative of the EIC environment, especially in the presence of hydrophobic chains. The potential energy surfaces for each pathway are plotted in Figure 6; full details are provided in the Supplemental Information. From Figure 6, it is clear that the most energetically favorable process is the N(α)-mediated pathway, which leads to a substantial lowering of the barrier (25 kcal mol−1) in comparison with the corresponding uncatalyzed process. In contrast, the imidazole pathway is much higher in energy and only slightly more favorable (by 6.4 kcal mol−1) than the uncatalyzed process. The lack of significant catalysis by the imidazole pathway can be explained by the absence of a H bond between the N(ɛ) atom of the imidazole ring of EIC and its carboxylate group, which mimics the Asp-His interactions that are found to play significant roles in enzyme catalysis.33Fersht A.R. Sperling J. The charge relay system in chymotrypsin and chymotrypsinogen.J. Mol. Biol. 1973; 74: 137-149Crossref PubMed Scopus (88) Google Scholar, 34Warshel A. Energetics of enzyme catalysis.Proc. Natl. Acad. Sci. USA. 1978; 75: 5250-5254Crossref PubMed Scopus (433) Google Scholar, 35Craik C.S. Roczniak S. Largman C. Rutter W.J. The catalytic role of the active site aspartic acid in serine proteases.Science. 1987; 237: 909-913Crossref PubMed Scopus (285) Google Scholar, 36Szeltner Z. Rea D. Juhász T. Renner V. Mucsi Z. Orosz G. Fülöp V. Polgár L. Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase.J. Biol. Chem. 2002; 277: 44597-44605Crossref PubMed Scopus (28) Google Scholar Moreover, the anionic carbonyl oxygen atom (C–O−) in the intermediate structure of the EIC (INT1 in Figure 4) is unstable and abstracts the proton back from the imidazole ring in the catalyst, resulting in a neutral (i.e., C–OH) tetrahedral intermediate in the ground state as opposed to the oxyanion intermediate found in the natural enzymatic system. The absence of a favorable electrostatic environment, as seen in the enzymes, can lead to instability of the oxyanion in the EIC-substrate intermediate (i.e., INT1), which in turn is stabilized after H is extracted from the imidazole unit. As noted above, the steric strain incurred in accommodating the catalytic triad mimicking hydroxyl-imidazole-carboxyl interactions also inflates the reaction barriers by this process. Modeling of the hydrophobically modified resins provides support for the formation of a hydrophobic pocket surrounding the EIC, analogous to the binding pocket of many native proteases (Figures S3 and S4). Simulations showed that the C16 hydrophobic residues can form a number of different contacts with the EIC across all of the examined fragments in which the modified residues were separated by zero to five styrene units (Figure 5C). In general, the C16 hydrophobic chain adopted a folded conformation, reflected in the consistent head-to-tail distance measured for this residue (Figure S3). The distance between the center of mass of the EIC and C16 hydrophobic residues on the resin support averaged approximately 10 Å, and distances as low as 1 Å were observed for some conformations where the C16 hydrophobic chain enfolded the EIC. The strong interactions between the EIC and C16 hydrophobic residues provide support for the formation of a local hydrophobic environment on the resin that can both attract substrate and tune the electrostatic interactions between EIC functional groups. These findings further support the experimental observations of balancing hydrophobic and catalytic residue inclusion on the resins for optimal catalysis. An interesting consequence of these studies is the indication that esterolysis by the EIC is conducted by two structural groups, the O(γ)H hydroxyl and the N(α)H secondary amine, which, as noted above, resemble the Ser-Lys catalytic dyads in some native protease enzymes.32Ekici O.D. Paetzel M. Dalbey R.E. Unconventional serine/threonine proteases: variations on the catalytic Ser/His/Asp triad configuration.Protein Sci. 2008; 17: 2023-2037Crossref PubMed Scopus (218) Google Scholar, 33Fersht A.R. Sperling J. The charge relay system in chymotrypsin and chymotrypsinogen.J. Mol. Biol. 1973; 74: 137-149Crossref PubMed Scopus (88) Google Scholar However, the other functional groups in the EIC, carboxylate and imidazole, are involved in a supporting role to stabilize the transition state and intermediate structures. This result is supported experimentally by the control structures (Figure 2D), which indicate that all three groups of the EIC are required for catalysis. The consequence of specific functional-group placement to best leverage the observed catalytic mechanism is of considerable interest, and this strategy holds promise for future bio-inspired catalyst designs. We have developed a simple one-step strategy for preparing multi-functional catalytic building blocks with a triad of reactive groups—carboxylate, imidazole, and hydroxyl—for CuAAC-mediated attachment to a variety of substrates. The robust and quantitative nature of the CuAAC reaction also allows the stepwise introduction of building blocks (C16 + triad) to be developed, leading to a series of polymer-supported EICs with finely tuned local hydrophobic environments. Significantly, the incorporation of C16 alkyl chains results in a 50-fold faster initial rate of reaction for the dual (C16 + triad) systems than for the parent triad functional resin. QM calculations illustrate a modified mechanism in comparison with native serine proteases, closely resembling the catalytic mechanism of Ser-Lys catalytic dyads. Both calculations and experimental results indicate that all functional groups within the EIC building block play an important role in the catalytic mechanism. Further work is underway to optimize the spatial arrangement of the functional moieties and nearby chemical environment to better harness the observed catalytic mechanism.