Kinetics of suicide substrates

Waley, S. G.

doi:10.1042/bj1850771

Cited by 161 publications

(83 citation statements)

References 10 publications

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“…preincubation of the enzyme in a small volume, followed by dilution of the mixture to the standard volume for an assay of the remaining activity, is a suitable procedure for the detection and study of enzyme-activated irreversible inhibitors (Waley, 1980). The 5-fold increase in reaction volume minimizes the effects of the inhibitor during the enzyme assay, and any remaining inactivation or competitive antagonism is accounted for by the addition of appropriate amounts of the inhibitor at the beginning of the assay period.…”

Section: Discussionmentioning

confidence: 99%

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

Günzler¹,

Hanauske-Abel²,

Myllylä³

et al. 1987

Biochemical Journal

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From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mm and the bimolecular rate constant 0.09 M-1 -s-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the a-subunit, but the f-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the a-subunit.

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Section: Discussionmentioning

confidence: 99%

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

Günzler¹,

Hanauske-Abel²,

Myllylä³

et al. 1987

Biochemical Journal

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“…Their metabolites were analyzed as described above. Kinetic parameters of inactivation process were calculated according to the method of Waley (1980Waley ( , 1985. The observed rate constant of inactivation (k obs ) was calculated from the initial slopes of the liner regression line of the "residual activity" versus "preincubation time" profile plotted on a semilogarithmic scale.…”

Section: Methodsmentioning

confidence: 99%

Metabolism of Sesamin by Cytochrome P450 in Human Liver Microsomes

Yasuda

Ikushiro²,

Kawamura³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k cat /K m value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

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“…1 shows the standard TDI kinetic model, in which the enzyme-inhibitor (EI) complex is converted to a reactive intermediate (EI*) which can either form an inhibitor metabolite (P I ) or inactivate the enzyme (E*) (Waley, 1980;Waley, 1985;Mohutsky and Hall, 2014). The equations derived with this scheme are as follows (Kitz and Wilson, 1962;Jung and Metcalf, 1975;Waley, 1980;Waley, 1985):…”

Section: Theoreticalmentioning

confidence: 99%

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 1. Theoretical Considerations

Nagar¹,

Jones²,

Korzekwa³

2014

Drug Metab Dispos

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Inhibition of cytochromes P450 by time-dependent inhibitors (TDI) is a major cause of clinical drug-drug interactions. It is often difficult to predict in vivo drug interactions based on in vitro TDI data. In part 1 of these manuscripts, we describe a numerical method that can directly estimate TDI parameters for a number of kinetic schemes. Datasets were simulated for Michaelis-Menten (MM) and several atypical kinetic schemes. Ordinary differential equations were solved directly to parameterize kinetic constants. For MM kinetics, much better estimates of K I can be obtained with the numerical method, and even IC 50 shift data can provide meaningful estimates of TDI kinetic parameters. The standard replot method can be modified to fit non-MM data, but normal experimental error precludes this approach. Non-MM kinetic schemes can be easily incorporated into the numerical method, and the numerical method consistently predicts the correct model at errors of 10% or less. Quasi-irreversible inactivation and partial inactivation can be modeled easily with the numerical method. The utility of the numerical method for the analyses of experimental TDI data is provided in our companion manuscript in this issue of Drug Metabolism and Disposition (Korzekwa et al., 2014b).

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Kinetics of suicide substrates

Cited by 161 publications

References 10 publications

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

Metabolism of Sesamin by Cytochrome P450 in Human Liver Microsomes

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 1. Theoretical Considerations

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