The Esterase Activity of Bovine Mercaptalbumin

Abstract

Abstract The results of a kinetic investigation of the reactions of bovine serum mercaptalbumin with p-nitrophenyl acetate are described. At pH 6.3 to 7.8 these reactions are biphasic and consist of (a) an initial phase during which the release of p-nitrophenol results from a monoacetylation of the albumin and from an albumin-catalyzed hydrolysis of the p-nitrophenylacetate and (b) a second phase during which the release of p-nitrophenol results only from the albumin-catalyzed hydrolysis of the ester. Second order rate constants for both the acetylation reaction and albumin-catalyzed hydrolytic reaction are reported. Results are also presented which indicate that the acetylation site and the catalytic site on the albumin are separate sites and that the conformational integrity of the protein is necessary for its catalytic activity. Analysis of albumin which has reacted with p-nitrophenyl acetate indicate that two tyrosyl hydroxyl groups on the protein are rapidly acetylated by the ester. One of these tyrosyl groups is at the acetylation site of the protein and the other is postulated to be adjacent to the catalytic site. A model compatible with the kinetic and analytical data is proposed in which the acetyl group of an acetyl protein intermediate in the catalytic reaction is transferred to a tyrosyl residue adjacent to the catalytic site.