Chicken serum albumin hydrolyzes dichlorophenyl phosphoramidates by a mechanism based on transient phosphorylation

The hydrolyzing activities of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP, and p-nitrophenyl butyrate (p-NPB) in chicken serum had been found t o copurify in the same protein, identified as albumin, The hydrolyzing acti vities of both chicken ser um and commercial serum albumins from different species were inhibited in a dose-dependent manner by short chain fatty acid s. On simultaneous incubation of chicken serum with HDCP and p-NPB, a compe titive interaction was detected between the two substrates, This behavior s uggests that both are hydrolyzed in the same albumin active site. When chic ken serum was preincubated with one of the substrates, and the latter were withdrawn by large dilution, the hydrolyzing activities with both substrate s were found to be reduced. This reduction was in turn dependent upon the t ime of preincubation with the first substrate. These results suggest that H DCP and p-NPB are hydrolyzed by the same albumin active site, via a mechani sm based on transient phosphorylation/acylation of the active site. The pro posed hydrolysis mechanism would account for the hydrolytic kinetics of bot h substrates.