Copper Nanoclusters as an Effective Enzyme Inhibitor on the Activity Modulation of α-Chymotrypsin

The present work is undertaken with an objective to find out the suitability of CuNCs on the activity modulation of a model enzyme, α chymotrypsin (α-ChT). This work also aims to highlight the role of surface chemistry corresponding to CuNCs in modulating the activity of α-ChT. For this purpose, two different types of CuNCs having chemically different surface ligands, namely, cysteine (Cys) and tannic acid (TA), have been synthesized. Subsequently, the interaction of these CuNCs with α-ChT has been investigated by employing various spectroscopic techniques at both ensemble average and single molecule level. Results obtained from enzyme kinetics studies have revealed that both the CuNCs act as good enzyme inhibitors. While Cys-CuNCs almost completely diminish the activity of α-ChT through a competitive inhibition mechanism, TA-CuNCs partially reduce the enzyme activity through a noncompetetive inhibition mechanism indicating the vital role of surface ligand in the regulation of α-ChT activity. To gain a molecular level understanding of the enzyme-inhibitor interaction event, fluorescence spectroscopy, ITC measurements, fluorescence correlation spectroscopy (FCS), agarose gel electrophoresis, and circular dichroism (CD) spectroscopy are conducted. Thermodynamics results obtained from fluorescence titration experiment and ITC measurements have indicated that Cys-CuNCs follow a one-step binding process, whereas TA-CuNCs follow a two-step binding process. Moreover, FCS studies have provided evidence for the interaction of CuNCs with α-ChT at the single molecule level. Importantly, circular dichroism (CD) measurements have demonstrated that the basic structure of α-ChT remains almost unaltered in the presence of CuNCs. The outcome of the present study is expected to open up a possibility of using CuNCs as an effective nanoscale enzyme regulator for various biological applications.