title: Refolding of a three (noncovalently linked)-domain enzyme. Human gamma-thrombin.
abstract: Human gamma-thrombin is a three (noncovalently linked)-domain enzyme which contains the known serine protease catalytic triad, Asp-His-Ser, one on each of the three noncovalent domains (Asp 99 on the A-B3 chain). While protein-folding dogma does not necessarily predict that the denatured form of this enzyme could refold to the correct conformation, a monitor of the esterase activity (Tos-Arg-OMe) shows complete recovery of native catalytically active conformation. When compared with the covalently intact alpha form which refolds from urea in less than 2 min with complete return of both clotting and esterase activity, gamma-thrombin requires up to 90 min to regain full esterase activity. The gamma-thrombin reactivation data best fit a single first order rate constant, k = 0.03 +/- 0.005 min-1. It was suggested that the gamma-thrombin renaturation process might represent first the rapid refolding, then subsequent reassociation and reisomerization of the three noncovalent domains to yield a lower energy, fully active, conformation. This study represents the only example known of the refolding (reconstitution) of a three (noncovalent)-domain protein.
mesh_terms: D001777:Blood Coagulation; D004950:Esterases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014508:Urea
chemical_list: D014508:Urea; D004950:Esterases; D013917:Thrombin