Zinc ions and zinc-embedded carbon quantum dots as competitive inhibitors of fumarase: preferential inhibition of the reverse reaction

Abstract

Targeted modulation of enzyme activity offers a promising strategy for both elucidating catalytic mechanisms and developing novel therapeutics. In this study Zn²⁺ ions were introduced as an effective competitive inhibitor of fumarase, a pivotal enzyme in the citric acid cycle. Zn²⁺ binding significantly alters the Michaelis constant (K_m) for both L-malate and fumarate, with a pronounced preference for inhibiting the reverse reaction (L-malate to fumarate), a direction relevant to redox homeostasis and anaplerotic flux. A major limitation of the clinical application of many metal-based inhibitors is their poor water solubility. To overcome this challenge and introduce a new class of enzyme inhibitors, zinc-modified carbon quantum dots (Zn–CQDs) were synthesized. Owing to their polar surface, Zn–CQDs interact more effectively with the enzyme, which increases the local concentration of Zn²⁺ ions at the active site. As a result, these nanomaterials exhibit enhanced water solubility and significantly greater inhibitory potency compared to free Zn²⁺ ions. Biophysical and kinetic analyses confirmed the competitive inhibition mechanism and demonstrated that Zn–CQDs interact with the enzyme without perturbing its secondary structure. Notably, both Zn²⁺ ions and Zn–CQDs preferentially inhibited the reverse reaction of fumarase, offering precise control over fumarase activity. Molecular docking and MD simulations elucidated the plausible binding site of Zn²⁺ within the active site. It was found that Zn²⁺ interacts with Glu340, a residue previously shown to be involved in binding fumarase inhibitors. These findings establish Zn–CQDs as a novel class of water-soluble fumarase inhibitors, distinguished by their facile synthesis, tunable solubility, and selective inhibition profile. This work highlights the potential of zinc-based nanomaterials in enzyme regulation, offering a powerful alternative to existing inhibitors and developing targeted redox-sensitive therapeutic strategies.