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: Under all the experimental conditions, values of K(s) (= k(-1)/k(+1)), k(+2), and k(+2)/K(s) 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)∼0s(-1). The pH dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a)-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.
Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin.
FASANO, MAURO
2012-01-01
Abstract
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: Under all the experimental conditions, values of K(s) (= k(-1)/k(+1)), k(+2), and k(+2)/K(s) 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)∼0s(-1). The pH dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a)-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.
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