Enzymes are an important
class of biomacromolecules which catalyze
many metabolic processes in living systems. Nanomaterials can be synthesized
with tailored sizes as well as desired surface modifications, thus
acting as promising enzyme regulators. Fluorescent gold nanoclusters
(AuNCs) are a representative class of ultrasmall nanoparticles (USNPs)
with sizes of ∼2 nm, smaller than most of proteins including
enzymes. In this work, we chose α-chymotrypsin (ChT) and AuNCs
as the model system. Activity assays and inhibition kinetics studies
showed that dihydrolipoic acid (DHLA)-coated AuNCs (DHLA-AuNCs) had
a high inhibitory potency (IC
50 = 3.4
μM) and high inhibitory efficacy (>80%) on ChT activity through
noncompetitive inhibition mechanism. In distinct contrast, glutathione
(GSH)-coated AuNCs (GSH-AuNCs) had no significant inhibition effects.
Fluorescence spectroscopy, agarose gel electrophoresis and circular
dichroism (CD) spectroscopy were conducted to explore the underlying
mechanisms. A two-step interaction model was proposed. First, both
DHLA-AuNCs and GSH-AuNCs might be bound to the positively charged
sites of ChT through electrostatic forces. Second, further hydrophobic
interactions occurred between three tyrosine residues of ChT and the
hydrophobic carbon chain of DHLA, leading to a significant structural
change thus to deactivate ChT on the allosteric site. On the contrary,
no such interactions occurred with GSH of zwitterionic characteristic,
which explained no inhibitory effect of GSH-AuNCs on ChT. To the best
of our knowledge, this is the first example of the allosteric inhibition
of ChT by nano regulators. These findings provide a fundamental basis
for the design and development of nano regulators.
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