Role of Arg-410 and Tyr-411 in human serum albumin for ligand binding and esterase-like activity

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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411

“…Site-directed mutagenesis experiments have shown that albumin esterase activity is abolished when Tyr 411 is mutated to Ala, and severely diminished when Arg 410 is mutated to Ala (Watanabe et al, 2000). These results support Tyr 411 as the active site for albumin esterase activity and support a role for Arg 410 in stabilizing the reactive anionic form of Tyr 411.…”

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

“…Subdomain IIIA of HSA possesses a well-known esteraselike activity toward substrates such as p-nitrophenyl acetate 12) and several N-carbobenzoxy-D(L)-alanine p-nitrophenyl esters. 113) Residues in this part of HSA are probably also able to detoxify cyanide by reaction with elemental sulfur to form thiocyanate.…”

“…(iii) Single-residue mutations in this region of albumin have a significant effect on the conformational and thermal stability of the protein, much more than mutations in site II. 12,13) Thus site I seems to be capacious and flexible and to have a large number of individual ligand-binding sites that sometimes are independent of each other but in other cases influence each other mutually. To characterize the site in more detail, several attempts to subdivide the site have been made.…”

“…However, investigations performed with rHSA mutants showed that 410 Arg is not important for diazepam binding. 12) 3-1-3. Additional Sites Although the site I and site II classification of Sudlow et al 6) remains useful, it cannot account for high-affinity binding of all drugs.…”

“…15) The hydrolytic activity toward p-nitrophenyl acetate has been studied in detail using several rHSA mutants including 411 Ser, and the results indicate that the presence of a hydroxy group in position 411 and the nature of its immediate surroundings are crucial for the esterase activity of HSA. 12) The enzyme-like properties of HSA can be of practical use. Thus a sensitive kinetic method for quantitating the protein utilizing its hydrolytic activity has been developed using Binding of two TIB molecules to crystals of rHSA complexed with five Myr (two in subdomain IIIA).…”