Spectroscopic techniques are powerful biophysical

Spectroscopic techniques are powerful biophysical tools used in the study of biomolecular structures, including those of proteins. UV–Vis enos inhibitor spectroscopy makes use of absorption property of the protein peptide backbone at around 218 nm, and aromatic amino acid residues primarily at around 280 nm. Changes in the absorption property at these wavelengths due to change in the environment of aromatic amino acid residues or orientation of peptide backbone structure enables this technique to sense any structural or conformational changes in proteins [24]. The intrinsic fluorescence of proteins is due to the aromatic amino acid residues, and is very sensitive to changes in the environment around these residues. Any such changes in the intrinsic fluorescence brought about by a quencher or a ligand molecule gives an idea of conformational changes within the protein molecules. When fluorescence quenching takes place due to fluorescence resonance energy transfer (FRET), the information about distance between fluorophore and quencher can be obtained [25,26]. CD spectroscopy makes use of differential absorption of left and right circularly polarized light by protein molecules to study the secondary and tertiary structure of proteins. Any changes in the secondary and tertiary structural element can be distinguished and measured using far and near UV CD spectroscopy [27,28]. Molecular docking is an important bioinformatics tool to study and predict the protein-ligand interactions. It is widely utilized in protein-ligand interaction studies for the prediction of ligand binding sites, binding residues, binding energy, type of binding forces, etc. [29].
Materials and methods
Results and discussion
Conclusions Caffeine was found to inhibit both the dehydrogenase and esterase activity of hsALDH. It reduced the substrate binding affinity and the catalytic efficiency of the enzyme. The mode of inhibition is mixed type with partial competitive and non-competitive behavior. Caffeine increased the pKa value of the enzyme and hence, the nucleophilicity of the catalytic Cys residue is reduced. It partially altered the secondary structure of the enzyme. Biophysical investigation revealed that caffeine forms a complex with hsALDH in a static manner. Molecular docking analysis revealed that caffeine binds to the active site of the enzyme and interacts with some of the highly conserved amino acid residues through non-covalent interactions. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing the substrate binding affinity and partially competing with the substrate to bind to the active site. Also, it reduces the catalytic activity of the enzyme by decreasing the nucleophilicity of catalytic Cys residue, and partially altering the secondary structure of the enzyme. The study indicates that oral intake of caffeine through different means may have a harmful effect on the oral health and may increase the risk of oral carcinogenesis. ALDH3A1 enzyme is occasionally over expressed in neoplastic tissues [11], resulting in increased resistance to oxazaphosphorine chemotherapy [12]. Therefore, it can be speculated that a cocktail of chemotherapeutic drug with ALDH3A1 inhibitors such as caffeine may act as an adjuvant and could increase the sensitivity of these drugs through its inhibitory effect on the enzyme.
Conflict of interest
Acknowledgements
Introduction Heart failure is associated with lipid peroxidation and the accumulation of α, β-unsaturated aldehydes [1], [2], which react avidly with cellular nucleophiles and can propagate or amplify tissue injury [3]. Although the heart possesses several aldehyde-detoxifying enzymes [4], [5], its capacity to remove aldehydes is diminished in heart failure. For example, aldose reductase, which reduces reactive aldehydes such as 4-hydroxynonenal (HNE) to relatively inert alcohols, is downregulated in the failing mouse heart [6], and glutathione-S-transferase activity, which conjugates aldehydes to glutathione, is lower in the pressure-overloaded rat heart [7]. Furthermore, the activity or abundance of aldehyde dehydrogenase 2 (ALDH2)—responsible for the majority of HNE detoxification in the heart [4], [5]—is diminished in ischemia [4], after myocardial infarction [8], [9], and in pressure overload-induced hypertrophy [10].