Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate.
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 (0.075 mM). 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: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. 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 (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), 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.
Masson
Patrick
P
Centre de Recherches du Service de Santé des Armées, Unité d'Enzymologie, BP 87, 38702 La Tronche Cedex, France. pymasson@compuserve.com
Froment
Marie-Thérèse
MT
Darvesh
Sultan
S
Schopfer
Lawrence M
LM
Lockridge
Oksana
O
D000083:Acetanilides / Q000737:chemistry*; D000418:Albumins / Q000737:chemistry; D000581:Amidohydrolases / Q000378:metabolism*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000737:chemistry; D005461:Fluorine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009584:Nitrogen / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D000083:Acetanilides; D000418:Albumins; D005227:Fatty Acids; D012709:Serum Albumin; D005461:Fluorine; D000581:Amidohydrolases; C019732:aryl acylamidase; D009584:Nitrogen; C508827:acetanilide