Investigation of the effect of homocysteine on the activity of carbonic anhydrase I and II isoenzymes

Homocysteine (Hcy) is a non-essential amino acid formed through the intracellular demethylation of methionine. Accumulation of Hcy at toxic levels (hyperhomocysteinemia, HHcy) has been associated with various pathological conditions such as atherosclerosis, cancer, Alzheimer's disease, glaucoma, and anemia. HHcy is also known to be linked to abnormal activities of carbonic anhydrase (CA) isoenzymes in many diseases. CA enzymes play a critical role in pH regulation, electrolyte homeostasis, and various biosynthetic processes by catalyzing the reversible hydration of carbon dioxide. In this study, the effects of Hcy on the activity of human CA I and CA II (hCA I and hCA II) isoenzymes were investigated through both in vitro analyses (using esterase and hydrase activity assays) and molecular docking simulations. L-histidine (HIS) and acetazolamide (AZM) were used as positive controls. DL-Hcy was prepared and tested at various concentrations ranging from 0 to 4000 µM. Results from both activity assays showed that DL-Hcy increased the activity of both isoenzymes. According to esterase activity, the AC50 values of DL-Hcy were calculated as 16.65 µM for hCA I and 23.32 µM for hCA II. Hydrase activity results showed AC50 values of 8.95 µM for hCA I and 55.91 µM for hCA II. In molecular docking analyses, crystal structures of CA I (PDB ID: 2foy) and CA II (PDB ID: 2fou) were used to simulate the binding of DL-Hcy, HIS, and AZM ligands and to evaluate their binding affinities. The analyses revealed that DL-Hcy is a weak inhibitor of CA II isoenzyme and exhibits low binding stability. In conclusion, when the AC50 values obtained from in vitro analyses are evaluated together with the molecular docking findings, it is concluded that DL-Hcy is a potential activator candidate that can enhance the activity of hCA I and hCA II isoenzymes.