Theory of the Transient Phase in an Enzyme System Involving Two Enzyme-Substrate Complexes: The Case of the Formation of Products From the First Complex

Ouellet, Ludovic; Stewart, James

doi:10.1139/v59-100

Cited by 38 publications

(4 citation statements)

References 7 publications

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“…The initial "burst" production of SN-38 is consistent with a deacylation-limited reaction (Maguire et al 1974;Ouellet and Stewart 1959). This effect has been shown for CPT-11 with a rat serum hydrolase (Tsuji et al 1991) and, more recently, a human liver microsomal carboxylesterase (Rivory et al 1996).…”

Section: Discussionsupporting

confidence: 64%

“…The Yaxis intercept (π) was also obtained (nM). For enzyme deacylation-limited reactions, the reaction velocity (ν) is given by (Ouellet and Stewart 1959):…”

Section: Methodsmentioning

confidence: 99%

“…where E 0 is the total enzyme concentration (Ouellet and Stewart 1959). Equation (1) was fitted to the reaction velocity data by nonlinear least-squares regression (SigmaPlot, Jandel) to obtain estimates of V max and K' m .…”

Section: Methodsmentioning

confidence: 99%

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The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes Differential hydrolysis for the lactone and carboxylate forms

Haaz

Rivory

Riche

et al. 1997

Naunyn-Schmiedeberg's Arch Pharmacol

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Irinotecan (CPT-11) is a new camptothecine derivative presently in development for the treatment of several advanced malignancies. It is converted in vivo to a highly potent metabolite, SN-38, by carboxylesterases. All camptothecine derivatives undergo lactonolysis in a pH-dependent reversible manner, generating inactive carboxylate forms. We have investigated in vitro the kinetics of transformation of CPT-11 to SN-38 by human liver microsomes originating from several donors. Microsomes from seven livers were studied individually or as a pooled preparation. CPT-11, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 formed was measured by HPLC with fluorometric detection. In the deacylation-limited carboxylesterase reaction, the linear steady-state kinetics between 10 and 60 min were determined. At all concentrations of CPT-11, the steady-state velocity of SN-38 formation as well as the intercept concentrations of SN-38 were about 2-fold higher when the substrate was under the lactone form than under the carboxylate form. We estimated the values (+/-SD) of K'm and Vmax to be 23.3 +/- 5.3 microM and 1.43 +/- 0.15 pmol/min/mg for the lactone and 48.9 +/- 5.5 microM and 1.09 +/- 0.06 pmol/min/mg for the carboxylate form of CPT-11, respectively. We conclude that the greater rate of conversion of CPT-11 lactone may contribute to the plasma predominance of SN-38 lactone observed in vivo. The inter-individual variation of SN-38 formation was relatively high (ratio of 4 between extreme values) but no large age- or gender-related differences were seen. The effect of twelve drugs of different therapeutic classes (antibiotics, antiemetics, antineoplastics, antidiarrhoeics, analgesics), which could be administered in association with irinotecan in the clinical setting, was evaluated in this system (drug concentration: 100 microM; CPT-11 lactone concentration: 10 microM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38.

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Section: Discussionsupporting

confidence: 64%

“…The Yaxis intercept (π) was also obtained (nM). For enzyme deacylation-limited reactions, the reaction velocity (ν) is given by (Ouellet and Stewart 1959):…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes Differential hydrolysis for the lactone and carboxylate forms

Haaz

Rivory

Riche

et al. 1997

Naunyn-Schmiedeberg's Arch Pharmacol

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“…the size of the 'burst' to the enzyme concentration indicates that k2 9 k , and that K ,< [S]O[31]. The slower disappearance of N-acetylbenzotriazole corrected for the spontaneous hydrolysis of the reagent corresponds to the turn-over for I Evolution of active-sites conceniration versus time of' incubation ut p H 8 5 .…”

mentioning

confidence: 99%

N-Acetylbenzotriazole as a Protein Reagent. Specific Behaviour towards delta-Chymotrypsin

Reboud‐Ravaux

Ghélis

1976

Eur J Biochem

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When N-['4C]acetylbenzotriazole, presented here as a new agent for the acetylation of proteins, reacted at pH 8 and 25 "C with 6-chymotrypsin, 15 amino groups (the e-amino groups of lysine residues and the a-amino terminus of half-cystine-1) and two phenolic groups (those of the two exposed tyrosine residues) were acetylated with respective pseudo first-order constants of 0.056 f 0.003 and 0.15 A 0.03 min-l. Surprisingly, in contrast with the acetic anhydride reaction, the a-amino group of Ile-16 was found to be not acetylated as shown by N-terminus determination and activity measurements : the modified d-chymotrypsin (or acetylated 6-chymotrypsin) was fully active after neutral dialysis. Only a transient inactivation due to the incorporation of one [14C]acetyl group per mole of catalytic site was observed. The kinetic constant found for reactivation at pH 8.5 was 0.315 k 0.005 min-' at 25 "C. The enzyme-catalyzed hydrolysis of N-acetylbenzotriazole was described by a k,,, value of 0.093 ? 0.005 min-' at pH 7 and 25 "C.Circular dichroism changes observed at 230 nm during the reaction at pH 8.5, of acetylated 6-chymotrypsin with N-acetylbenzotriazole indicated a total conversion of the amount of enzyme molecules which were in the 'inactive' or 'alkaline' conformation at this pH, into the 'active' or 'neutral' one. Benzotriazole alone was unable to induce such a conformational change. The rate constant of the reverse structural process from the 'neutral' to the 'alkaline' conformation was 0.32 k 0.02 min-' : identical to that of the deacetylation of the catalytic site. Thus, the unusual lack of acetylation of Ile-16 a-amino group during 6-chymotrypsin treatment with N-acetylbenzotriazole is interpreted as a stabilization of the enzyme 'neutral' conformation where the Ile-16 m-amino group is buried, thus inaccessible to the reagent.The properties of the S-chymotrypsin modification using N-acetylbenzotriazole led to practical uses : direct spectrophotometric titration of chymotrypsin operational normality at pH 7 and rapid preparation of acetylated 6-chymotrypsin. As a protein reagent, N-acetylbenzotriazole is particularly interesting because of its reactivity towards amino and phenolic groups of amino acid residues, its stability at acid pH, i.e., khydrolysls = 7.38 x min-l at 25 "C [Ravaux et al. (1971) Tetrahedron Letters, 4013 -401 51 and its aromaticity, responsible for optical properties.

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Kinetics of ‘initial burst’ in the solid–liquid phase‐transfer catalysis. Nucleophilic substitution of 2‐octyl mesylate with potassium bromide

Yufit

Zinovyev

2001

J of Physical Organic Chem

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The S N 2 substitution of 2-octyl mesylate with solid KBr under the conditions of phase-transfer catalysis was studied kinetically using the model approach of 'initial burst.' It is suggested that such kinetics reflect the contribution of mass transfer and surface poisoning. The proposed model is used to explain the influences of catalyst, solvent, stirring speed, activation and agitation effects. The mechanistic scheme suggests that the reaction is described by two separate kinetic stages, one of which reflects the intrinsic rate-limited step and the other the mass transfercontrolled step.

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Theory of the Transient Phase in an Enzyme System Involving Two Enzyme-Substrate Complexes: The Case of the Formation of Products From the First Complex

Cited by 38 publications

References 7 publications

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes Differential hydrolysis for the lactone and carboxylate forms

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes Differential hydrolysis for the lactone and carboxylate forms

N-Acetylbenzotriazole as a Protein Reagent. Specific Behaviour towards delta-Chymotrypsin

Kinetics of ‘initial burst’ in the solid–liquid phase‐transfer catalysis. Nucleophilic substitution of 2‐octyl mesylate with potassium bromide

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