Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin

Most of the esterase properties of human serum albumin (HSA) are the result of multiple irreversible chemical modifications rather than turnover. The HSA-catalyzed hydrolysis of 4-nitrophenyl myristate (NphOMy) is consistent with the minimum three-step mechanism involving the acyl-enzyme intermediate HSA-OMy: HSA+NphOMy⇄k-1k+1HSA:NphOMy⇄k-2k+2HSA-OMy+NphOH⇄k-3k+3HSA+MyOH Under all the experimental conditions, values of Ks (= k−1/k+1), k+2, and k+2/Ks determined under conditions where [HSA] ⩾ 5 × [NphOMy] and [NphOMy] ⩾ 5 × [HSA] match very well each other. The deacylation process is rate limiting in catalysis (i.e., k+3 ≪ k+2) and k−2 ∼ k−3 ∼ 0 s−1. The pH dependence of k+2/Ks, k+2, and Ks reflects the acidic pKa-shift of one ionizing group from 8.9 ± 0.2 in NphOMy-free HSA to 6.8 ± 0.3 in the HSA:NphOMy adduct. The HSA-catalyzed hydrolysis of NphOMy is inhibited competitively by diazepam, indicating that Tyr411 is the active-site nucleophile.

► Esterase activity of human serum albumin.
► Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin.
► Human serum albumin acylation mechanism.
► Diazepam inhibits human serum albumin acylation.
► Modulation of human serum albumin acylation by pH.