A linearization method for low catalytic activity enzyme kinetic analysis

Toti, Paolo; Petri, Antonella; Pelaia, Valerio; Osman, Azizah; Paolini, Moreno; Bauer, C.

doi:10.1016/j.bpc.2004.12.043

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…The method also requires knowledge of the active site residue that covalently binds the acyl group of the substrate during catalysis. The active site of human albumin for esterase activity is Tyr 411 [5,22,24]. Table I lists the sequence of the Tyrosine 411 peptic peptides of human albumin and their masses before and after covalent attachment of the acyl group from p-NPA and from o-NTFNAC.…”

Section: Maldi-tofmentioning

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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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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Section: Maldi-tofmentioning

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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“…In 1988 Segel [150] and in 1989 Segel and Slemrod [151] obtained the Michaelis-Menten approximation expanding the solutions in terms of a new parameter, including the so-called Michaelis constant and showing that the sQSSA is valid in a wider range of parameters than the one supposed before. However it is well known that while in vitro the condition on the concentrations can be easily fulfilled, in vivo it is not always respected [152,153,155,1,159,51], in particular when the reaction is not isolated but is part of complex reaction networks. This means that, though very useful, this approximation not always can be applied, at least in several situations of medical and pharmaceutical interest.…”

Section: Introductionmentioning

confidence: 99%

New trends and perspectives in nonlinear intracellular dynamics: one century from Michaelis–Menten paper

Bersani

Bersani²,

Dell’Acqua

et al. 2014

Continuum Mech. Thermodyn.

124

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One century after the seminal work by Leonor Michaelis and MaudMenten devoted to the theoretical study of the enzymatic reactions, in this paper we give an overview of the most recent trends concerning the mathematical modeling of several enzymatic mechanisms, focusing on its asymptotic analysis, which needs the use of advanced mathematical tools, such as Center Manifold Theory, Normal Forms, and Bifurcation Theory. Moreover we present some perspectives, linking the models here presented with similar models, arising from different research fields.

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“…For peroxidases (being two-substrate enzymes) these parameters can be determined when the H 2 O 2 concentration is kept at saturating concentrations (>5 mM) and the substrate concentration is varied. 49, 50 Determining the kinetic parameters indeed showed that the encapsulated enzyme displayed the same saturation kinetics as the free enzyme and the K M for thioanisole appeared to be similar (Table 2 and Fig. S6 †).…”

Section: Sulfoxidation Inside Cpsmentioning

confidence: 81%

Biocatalytic oxidation by chloroperoxidase from Caldariomyces fumago in polymersome nanoreactors

et al. 2009

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The encapsulation of chloroperoxidase from Caldariomyces fumago (CPO) in block copolymer polymersomes is reported. Fluorescence and electron microscopy show that when the encapsulating conditions favour self-assembly of the block copolymer, the enzyme is incorporated with concentrations that are 50 times higher than the enzyme concentration before encapsulation. The oxidation of two substrates by the encapsulated enzyme was studied: i) pyrogallol, a common substrate used to assay CPO enzymatic activity and ii) thioanisole, of which the product, (R)-methyl phenyl sulfoxide, is an important pharmaceutical intermediate. The CPO-loaded polymersomes showed distinct reactivity towards these substrates. While the oxidation of pyrogallol was limited by diffusion of the substrate into the polymersome, the rate-limiting step for the oxidation of thioansiole was the turnover by the enzyme.

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A linearization method for low catalytic activity enzyme kinetic analysis

Cited by 6 publications

References 24 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

New trends and perspectives in nonlinear intracellular dynamics: one century from Michaelis–Menten paper

Biocatalytic oxidation by chloroperoxidase from Caldariomyces fumago in polymersome nanoreactors

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