Abstract
Recombinant wild-type and mutant human serum albumins were produced using a yeast expression system. We investigated, by ultrafiltration, the high affinity binding of two representative site II-ligands, namely ketoprofen(KP) and diazepam(DZ). According to the crystal structure of HSA, the residues of 410Arg and 411Tyr protrude into the center of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of 410Arg the phenolic oxygen and the aromatic ring of 411Tyr are important for KP binding. The guanidino moiety probably interacts electrostatically with the carboxyl group of KP, the phenolic oxygen could make a hydrogen-bond to the ketogroup of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of KP. By contrast, 410Arg is not important for DZ binding. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that although 410Arg is important, the enzymatic activity of albumin is much more dependent on the presence of 411Tyr. In order to characterize the warfarin (WF) binding site, 199Lys, 214Trp, 218Arg and 242His were mutated. At pH 7.4, the affinity of H242Q and K199A were found to be about 6 and 4 times higher as compared to that of wild-type HSA, respectively. However, the affinity of R218H and W214A for WF were about 3 and 2 times reduced.These results show that the side chain of 214Trp and 218Arg are important for WF binding. In contrast, t he side chains of 199Lys and 242His interfere with binding. Finally, the effect of the N-B transition on WF binding will be discussed.
