ヒト血清アルブミン分子上の薬物結合部位のトポロジー解析

抄録

Human serum albumin (HSA), one of the most abundant proteins in the circulation system, contains binding sites for a variety of drugs. The recent X-ray crystallographic data of HSA shows that the two major ligand binding sites, site I and site II, are located within specialized cavities in two separate subdomains, namely subdomain IIA and IIIA, respectively. Correctly folded single-residue mutants K199A, W214A, R218H and H242Q were overexpressed. The primary binding constant for warfarin was decreased 2- and 3-fold by mutating Trp-214 and Arg-218, respectively, but was increased 4 and 6-fold by replacing Lys-199 and His-242, respectively. The constants for W214A and R218H responded to the pH-changes in a quantitatively different manner from that of K199A and H242Q. Thus, this parameter also showed heterogeneity in domain II in responding to the N to B transition. The single-mutants R410A, Y411A, Y411S and Y411F and the double-mutant R410A/Y411A were produced. We investigated, by ultrafiltration and CD, the high affinity binding of two representative site II-ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the Arg-410 and Tyr-411 residues protrude into the center of site II (in subdomain IIIA), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxyl group of ketoprofen, the phenolic oxygen could participate in hydrogen bonding to the ketogroup of the ligand, and the aromatic ring may participate in a specific stacking interaction with one or both of the aromatic rings of ketoprofen. In contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam and probably do so in principally the same way as with ketoprofen. 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 Arg-410 is important, the enzymatic activity of albumin is much more dependent on the presence of Tyr-411. Minor activity was detected when serine was introduced but not alanine or phenylalanine, in position 411.