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  • HsALDH enzyme has been purified for

    2024-05-18

    HsALDH enzyme has been purified for the first time in our laboratory from human saliva and has been kinetically characterized using different aromatic substrates [35]. Also, the effect of some common substances frequently encountered by the enzyme in the oral cavity (such as ethanol, hydrogen peroxide and sodium dodecyl sulfate), on its activity has been investigated [35]. More recently, the activity and stability of the purified enzyme was revealed under different conditions such as temperature, in presence of denaturants and salt [36]. Further, the storage stability of the enzyme was determined under in vitro conditions and in presence of stabilizing agents [36]. In one of our previous study, a small molecule chemical activator, Alda-1 was designed for ALDH2 which activated the enzyme and restored the activity of its inactive mutated form i.e., ALDH2*2 [11]. Recently, we have also reported sulforaphane, a natural molecule found in cruciferous vegetables as an activator of hsALDH, which increased the dehydrogenase the article activity of the enzyme by almost two fold [37]. The present study aimed at searching for a better natural molecule activator of hsALDH from various sources and found TQ as a suitable candidate. Therefore, we have studied the effect of TQ on the activity (dehydrogenase and esterase activity) and kinetics of both crude and purified hsALDH. The binding of TQ with the pure hsALDH was studied using different biophysical techniques such as UV–vis, fluorescence, CD spectroscopy and FRET analysis. Molecular docking was performed to determine the binding site, amino the article residues involved and the type of interactions between TQ and hsALDH. The interaction of TQ with hsALDH and its effect on the enzyme activity is expected to have a great importance from the point of view of aldehyde related pathogenesis, carcinogenesis and nutritional benefits on health.
    Materials and methods
    Results
    Discussion In one of our previous studies, we have reported a small molecule activator (Alda-1) of the wild type ALDH2 which also restored the activity of the inactive, mutated ALDH2*2 [11]. More recently, we have found sulforaphane from cruciferous vegetables as an activator of hsALDH, having significance in the metabolism of acetaldehyde [37]. It activated the enzyme by almost two fold. In the present study, we report the natural compound, TQ as a better activator of hsALDH, which increased the dehydrogenase activity of the enzyme by almost three fold. TQ may be used in mitigating aldehyde toxicity and maintenance of oral health. TQ activated both the crude and the pure hsALDH to a good extent with an EC50 value of 103.6±2.5nM and 109.8±2.3nM, respectively. The increase in the apparent Vmax value of the enzyme in the presence of TQ also shows that the catalytic activity of the enzyme is favored in the presence of TQ. The decrease in the Km value shows that TQ increases the affinity of the enzyme for the substrate. Also, the docking analysis depicted that TQ binds to the active site near the catalytic cysteine residue. Binding of TQ at the active site enhances the affinity of the enzyme for the substrate and does not alter the secondary structure of the enzyme upon binding. Therefore, these observations possibly suggest that TQ shows positive cooperativity in the binding of the substrates by the enzyme. TQ also promoted the esterase activity of the enzyme at nanomolar concentration although to a smaller extent. There was no significant change in the pKa value of hsALDH in the presence of TQ, and hence this implies that the nucleophilicity of the catalytic cysteine is not affected by TQ. Therefore, the increase in activity of the enzyme is not due to a change in the nucleophilicity of the catalytic cysteine. We speculate that the increase in the activity of the enzyme by TQ may be partly due to the increase in the affinity of the enzyme for the substrate, and because of the anti-oxidant property of TQ which protects the active site cysteine residue from oxidation and maintains it in the reduced form for catalysis to occur. The former likely happens due to the binding of TQ which may enable more favourable binding of the substrate to the active site, and optimise its proximity and orientation through local fitting of precise conformation of the substrate molecule, without altering the secondary structure of the enzyme.