On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases

Darvesh, Sultan; McDonald, Robert; Darvesh, Katherine Valenta; Mataija, Diane; Mothana, Sam; Cook, Holly; Carneiro, Karina M. M.; Richard, Nicole; Walsh, Ryan; Martin, Earl

doi:10.1016/j.bmc.2006.02.021

Cited by 39 publications

(39 citation statements)

References 36 publications

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“…The AAA activity of FAF-HSA was determined with o-NTFNAC as the substrate in 60 mM Tris/HCl buffer, pH 8.0 at 258C [10]. The substrate concentration varied from 0.005 to 3 mM.…”

Section: Measurement Of the Aaa Activity Of Albumin With O-ntfnacmentioning

confidence: 99%

“…Indeed, BuChE was found to be a possible contaminant of human albumin preparations [8] mostly because its monomer (C 1 ) forms a covalent conjugate with albumin (C 2 component in the multiple molecular form pattern of human plasma BuChE) [9]. In addition, BuChE displays a high AAA activity with o-NTFNAC [10]. However, in contrast to HSA, the FAF-HSA preparation was found to be free of BuChE based on highly sensitive cholinesterase activity detection on polyacrylamide gel.…”

Section: Absence Of Contaminating Enzymes In Faf-hsamentioning

confidence: 99%

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

Masson

Froment

Darvesh

et al. 2007

Journal of Enzyme Inhibition and Medicinal Chemistry

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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 ¼ 0.13^0.02 min2 1 and K s ¼ 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 ¼ k 2 ). Though the aryl acylamidase activity of albumin is low (k cat /K s ¼ 195 M 21 min 21 ), 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.

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“…The AAA activity of FAF-HSA was determined with o-NTFNAC as the substrate in 60 mM Tris/HCl buffer, pH 8.0 at 258C [10]. The substrate concentration varied from 0.005 to 3 mM.…”

Section: Measurement Of the Aaa Activity Of Albumin With O-ntfnacmentioning

confidence: 99%

Section: Absence Of Contaminating Enzymes In Faf-hsamentioning

confidence: 99%

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

Masson

Froment

Darvesh

et al. 2007

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…Whereas aryl-acylamidase activity in vertebrates usually (but not invariably) is associated with cholinesterases (14,23), which belong to the ␣/␤-hydrolase-fold superfamily of proteins, the assignment of most bacterial aryl-acylamidases to enzyme families is not known due to lack of sequence information. An exception is carbamate hydrolase from Arthrobacter oxydans P52, a 55-kDa protein with minor amidase activity, which indeed shows significant similarity to eukaryotic acetylcholinesterase and other large carboxylesterases (55).…”

Section: Discussionmentioning

confidence: 99%

“…In its preference towards o-nitroacetanilide, Amq functionally resembles human acetylcholinesterase rather than aryl-acylamidase from P. fluorescens, which hydrolyzes p-nitroacetanilide with higher levels of activity than the o-nitro analogue (14). 1 H-nuclear magnetic resonance analyses performed by Darvesh et al (14) suggested that o-nitroacetanilide forms a six-membered cyclic species involving intramolecular hydrogen bonding between the oxygen of the nitro substituent and the amide hydrogen. N-acetylanthranilate, the physiological substrate of Amq, may form an analogous intramolecular hydrogen bond (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The biological function of this additional reactivity of cholinesterases is not known; however, it has been discussed in several studies that it may be important in embryogenesis and early development of the nervous system and in pathological processes such as formation of neuritic plaques in Alzheimer's disease (5,12,14). Cholinesterases are thought to use the same active site, i.e., the same acyl binding pocket and the catalytic triad, for esterase and aryl-acylamidase activity (14).…”

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N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

et al. 2006

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N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

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Butyrylcholinesterase: Overview, Structure, and Function

Lockridge

Duysen

Masson

2011

Anticholinesterase Pesticides

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On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases

Cited by 39 publications

References 36 publications

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

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

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

Butyrylcholinesterase: Overview, Structure, and Function

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