Improving the catalytic efficiency of Fibrinolytic enzyme from Serratia marcescens subsp sakuensis by chemical modification

Microbial fibrinolytic enzymes have gained increased attention due to their potential to prevent or cure cardiovascular diseases. Promising natural enzymes are often modified to improve/enhance the kinetic constants. Hence an attempt was made to chemically modify the fibrinolytic enzyme produced by marine Serratia marcescens subsp. sakuensis using amino acid specific modifiers. The aim was to enhance the kinetic constants and gather information on the vital amino acid residues involved in the catalysis. Modification of cysteine, histidine, tryptophan and serine residues resulted in drastic reduction in fibrinolytic activity indicating their presence in the active site. Modification of carboxylate residues resulted in a 19-fold increase in specific activity suggesting their presence in the catalytic site. Interestingly, ratio of fibrinolytic to fibrinogenolytic activity of the modified enzyme did not change significantly. There was a 507-fold reduction in K-m value after chemical modification and due to that, 219-fold enhancement of catalytic efficiency was evidenced. Circular dichroism spectrum analysis of the modified and native enzyme revealed changes in alpha-helix and beta-sheet conformation of the enzyme. Furthermore, the modified enzyme was more responsive to the presence of most of the metal ions tested.