## 3 Results and Discussion
[**NP-Zn**] = [NINP] = 5.0 \(\upmu\)M. (**b**) Dependence \(V_{0}\) on the substrate concentration for the hydrolysis of **1c** and **1d** by **NP-Zn** in a 25 mM HEPES buffer (pH 7.0) containing 25 vol % methanol at 25 \({}^{\circ}\)C. [**NP-Zn**] = 5.0 \(\upmu\)M. The nanoparticles were prepared using 0.5 equiv of DVB. empty diamond data points). It should be mentioned that **1c** and **1d**, due to their higher hydrophobicity, are insoluble in water at high concentrations, and 25 vol % methanol was added to keep the reaction mixture homogeneous for the Michaelis-Menten experiments (Figure S7). In our current design, the template (**T**) resembles the desired substrates (e.g., **1b** and **1c**) so that the imprinted active site will have an affinity for the substrate (akin to the \(K_{\mathrm{M}}\) value in an enzymatic reaction). The affinity not only increases the effective concentration of the substrate near the catalytic groups but also positions the reactive group (i.e., the carbonyl) near the catalytic cofactor (i.e., the zinc atom). For a \"lock-and-key\" system, the shape of the active site is key to substrate selectivity. For the same reason, the current catalyst is not expected to hydrolyze a polymeric substrate such as BPA-PC because its long hydrophobic backbone cannot be accommodated by the active site of **NP-Zn**. Table 1 summarizes the Michaelis-Menten parameters for the catalytic hydrolysis of several carbonate substrates. The \(k_{\mathrm{cat}}\) value is the highest for **1b**, the compound with the best leaving group (entry 1). We also studied the hydrolysis of this compound in 25% methanol (entry 3) so that it can be compared directly with **1c** in the same solvent (entry 4). Compounds **1b** and **1c**, having a reasonably sized _para_-substituent on the phenyl rings, are both expected to fit within the active site of **NP-Zn** due to their similarity to the template in size and shape (Scheme 1). In 25% methanol, **1b** has a 3.2\(\times\) turnover number and \(\sim\)30% stronger binding than **1c**. In the end, the catalytic efficiency (\(k_{\mathrm{cat}}/K_{\mathrm{M}}\)) is four times higher for **1b** than for **1c**. When **1b** and **1e** are compared, the catalytic efficiency for the two substrates differs by a factor of 22, with positive contribution from both \(k_{\mathrm{cat}}\) (a factor of 3.3) and \(K_{\mathrm{M}}\) (a factor of 6.6). In other words, the dominant factor is the binding affinity for the better substrate. If we compare the hydrolysis of **1b** in water and 25% methanol, the \(k_{\mathrm{cat}}\) value decreases by 2.3 times, but the binding weakens by 6 times (Table 1, entries 1 and 3). The result confirms the hydrophobically driven binding of the substrate, as the addition of an organic solvent (methanol) to the reaction medium is expected to significantly reduce the driving force for the substrate to enter the active site. The hydrophobically driven binding is also supported by the weak binding of **1e** (entry 4). With only hydrogen on the _para_ positions, **1e** could only fill part of the active site. The situation is unfavorable whether the rest of the active site is left empty or filled with water molecules. The literature-reported rate constant (\(k_{\mathrm{uncat}}\)) for the uncatalyzed hydrolysis of diphenyl carbonate **1e** in water is 1.2 \(\times\) 10\({}^{-8}\) s\({}^{-1}\)[33]. Thus, **NP-Zn** is able to accelerate the hydrolysis by ca. 1 \(\times\) 10\({}^{6}\) at pH 7 and room temperature. A de novo designed thermostable polycarbonate hydrolase has an activity of 18.2 uM/(min\(\cdot\cdot\)mg) for **1a** at 25 \({}^{\circ}\)C in pH 8 buffer [16]. As mentioned above, this particular substrate reacts too fast under our experimental conditions. Nonetheless, **NP-Zn** is able to turn over the less reactive **1b** with an activity of 23.6 uM/(min\(\cdot\cdot\)mg) at 25 \({}^{\circ}\)C in pH 7 buffer. Thus, the activity is about 30% higher for a less reactive substrate under a less basic solution, underscoring the efficiency of our artificial nanozyme.