Homocysteine Thiolactone Modification of Ribonuclease A: Thermodynamics and Kinetics

Homocysteine thiolactone is a metabolite associated with various diseases at elevated levels in humans. Lysine residues in proteins are modified through N-homocysteinylation and homocysteinylated proteins are prone to form dimers and oligomers through disulfide cross-linkages. This study investigates the effects of N-homocysteinylation on Ribonuclease A (RNase A). The formation of dimers and higher oligomers in RNase A have been confirmed by SDS-PAGE and MALDI-ToF. Agarose-gel assays revealed an altered ribonucleolytic activity due to Lys modification. Fluorescence spectroscopy indicates local changes in the Tyr microenvironment. CD melting studies reveal that β-sheet formation is slightly enhanced with a reduction in the α-helical content in case of modified RNase A. However, the similar melting temperature of both native and modified RNase A indicates overall structural integrity with local changes in secondary structural components. ITC and UV-visible kinetics show reduced ribonucleolytic activity in homocysteinylated RNase A compared to the unmodified enzyme. These findings provide insights into the structural and functional consequences of RNase A homocysteinylation, contributing to our understanding of hyperhomocysteinemia-related pathologies.