Purpose: To elucidate the catalytic mechanism of the esterase-like activity of serum albumin (SA), the reactivity of SA from six species was investigated using p-nitrophenyl esters as model substrates.
Methods: The effect of pH and the energetic and thermodynamic profiles of SA were determined for all species for p-nitrophenyl acetate (PNPA). Then, kinetic and thermodynamic studies using a series of p- and o-nitrophenyl esters with different side chains and human SA (HSA) were carried out. The influence of deuterium oxide was also evaluated. Finally, the information gained was used to construct a computer model of the structural chemistry of the reaction.
Results: The pH profiles suggest that the nucleophilic character of the catalytic residue (Tyr-411 in the case of HSA) is essential for activity. This kcat-dependent activity was found to increase with a decrease in the activation free energy change (deltaG). Hence, the magnitude of deltaG, which is dependent on activation entropy change (deltaS), as calculated from the thermodynamic analysis, can be regarded as an indicator of hydrolytic activity. It indicates that p-nitrophenyl propionate (PNPP) is the best substrate by evaluating the reactions of nitrophenyl esters with HSA. The findings here indicate that deuterium oxide has no significant effect on the rate of hydrolysis of PNPA by HSA.
Conclusions: The results are consistent with a scenario in which HSA becomes acylated due to a nucleophilic attack by Tyr-411 on the substrate and then is deacylated by general acid or base catalysis with the participation of water.