Tuning the Catalytic Activity and Substrate Specificity of Peptide‐Nanoparticle Conjugates

Abstract

The immobilization of cysteine‐containing peptides onto the surface of gold nanoparticles (Pep‐Au‐MPCs) emerged as a promising strategy towards the development of artificial enzymes. In this context we studied the effect the location of the catalytic unit within the peptide‐monolayer relative to the nanoparticle surface has on the esterolytic activity and substrate specificity of three Pep‐Au‐MPCs, that only differ in the position of the catalytic unit (surface proximal, intermediate, surface distal). Rates of ester hydrolysis were found to correlate with the hydrophobicity of the substrate and the position of the catalytic unit. Highly hydrophobic ester substrates are cleaved more efficiently surface proximal, whereas less hydrophobic substrates showed higher rates of hydrolysis in the intermediate region of the monolayer. Our studies reveal the importance the position of the catalytic center has on the catalytic activity and substrate specificity of peptide‐nanoparticle conjugates.

Peptide–Gold Nanoparticle Conjugates as Artificial Carbonic Anhydrase Mimics

“…By applying supernatant analysis, the mean peptide loading per nanoparticle was determined to be 859 peptides/Au-NP. This corresponds to an average peptide density of 2.26 pep/nm 2 , which is consistent with literature reported peptide densities in the range of 1.41-2.55 pep/nm 2 , depending on the peptide length [9,11,22,23]. As the formation of β-sheets is a prerequisite for the generation of the 3-His Zn(II)-binding site, and thus the successful coordination of Zn(II) [19], circular dichroism (CD) spectroscopy was performed in order to assess the secondary structure of (un)conjugated IHQ-NP and its Zn(II)dependency.…”

“…While the dependence of unconjugated Zn(II)-/IHQ-NP-catalyzed 4-NPA hydrolysis on 4-NPA concentration displays hyperbolic behavior (Figure 3a), which is in accordance with the Michaelis-Menten theory, sigmoidal behavior was observed for Zn(II)-/Au@IHQ-NP ( Figure 3b). A sigmoidal profile indicates a positive cooperative effect for the binding of substrate molecules among individual active sites and has been previously observed to be an effect evoked by the conjugation and the resulting high peptide density within the peptide monolayer [9,11]. Positive cooperativity in substrate binding is classified as a regulatory mechanism also found in nature.…”

“…Thus, comparisons of catalytic efficiency should be done carefully. Interestingly, independent of the conjugate, all show a similar K M of approximately 0.5 mM [9][10][11], indicating a similar affinity of 4-NPA for the respective peptide monolayers.…”

“…The conjunction of peptides and Au-NPs leads to dense peptide monolayers in which new hydrogen bond and/or charge interactions between individual peptides are established. This enables known enzyme-like effects, such as the perturbation of pK a values of functional groups and cooperativity in substrate binding [9,11], and can even lead to new catalytic mechanisms [7,8]. Altogether, these effects can greatly improve the catalytic performance of a conjugated versus an unconjugated peptide.…”

“…To date, the design and application of Pep-Au-NPs have been limited to organic esterase chemistry, in which catalysis is mediated by a His-imidazole [7][8][9][10][11]. Considering the fact that zinc metalloenzymes perform numerous other types of reactions in nature, including phosphodiester and amide hydrolysis as well as the (de)hydrogenation of alcohols [12,13], it is of great interest to reconstitute such transformations in vitro by developing appropriate robust synthetic systems.…”