Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Patrick Masson, Marie Thérése Froment, Sultan Darvesh, Lawrence M. Schopfer, Oksana Lockridge

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: kcat =0.13 ± 0.02 min-1 and Ks=0.67 ± 0.04mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (kcat=k2). Though the aryl acylamidase activity of albumin is low (kcat/ Ks = 195M-1min-1), because of its high concentration in human plasma (0.6-1mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.

Original languageEnglish (US)
Pages (from-to)463-469
Number of pages7
JournalJournal of Enzyme Inhibition and Medicinal Chemistry
Volume22
Issue number4
DOIs
StatePublished - Aug 2007

Keywords

  • Albumin
  • Aromatic amide
  • Aryl acylamidase
  • High enzyme concentration
  • MALDI-TOF
  • Tyr 411

ASJC Scopus subject areas

  • Pharmacology
  • Drug Discovery

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