Abstract
Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. 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 residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), 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 carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in 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 enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411.