Kinetic analysis of protein modification reactions at equilibrium

Rakitzis, Emmanuel T.

doi:10.1042/bj2630855

Cited by 3 publications

(8 citation statements)

References 28 publications

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“…The analysis of the inactivation kinetics is consistent with the mechanism of reactions at equilibrium (Rakitzis, 1989). Several authors have studied the equilibrium between enzymes and modifying agents to produce covalently modified proteins, such as inactivation of 1980) and ornithine transcarbamylases (Marshall and Cohen, 1977) by pyridoxal-5'-phosphate, and glyceraldehyde-3phosphate dehydrogenase by butane-2,3-dione (Rakitzis, 1989). It is noteworthy that the reversion of a covalent modification is only possible in addition reactions (e.g.…”

Section: Discussionsupporting

confidence: 77%

“…-A,)]. been described that provide non-first-order kinetics: hydrolysis of the reagent (Perez-Gil et al, 1989); turnover on mechanismbased inhibitors (Waley, 1985); partially active modified enzyme species (Ray and Koshland, 1961); and modification reactions at equilibrium (Rakitzis, 1989).…”

Section: Resultsmentioning

confidence: 99%

“…the formation of a Schiff base from pyridoxal and lysine residues). Substitution reactions might occasionally be reversible, provided that the second product of the reaction is present in high enough concentrations (Rakitzis, 1989). As covalent modification of proteins by EEDQ implies the irreversible decomposition of reagent, as established above, the equilibrium state would never be reached.…”

Section: Discussionmentioning

confidence: 98%

“…Kinetics of enzyme modification reactions at equilibrium: correction for re-activation in the enzyme assay The kinetic formalism followed in this paper is based on the original model of Rakitzis (1989). The general scheme of protein modification reactions at equilibrium can be presented as the reversible interconversion of three species.…”

Section: Appendixmentioning

confidence: 99%

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Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

Martín

Mancheño

Arche

1993

Biochemical Journal

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Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.

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Section: Discussionsupporting

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 98%

Section: Appendixmentioning

confidence: 99%

See 2 more Smart Citations

Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

Martín

Mancheño

Arche

1993

Biochemical Journal

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“…Thermodynamic and kinetic analysis of protein modification and enzyme inactivation reactions and of regeneration by dilution of covalently modified proteins have been performed (34)(35)(36). Examples are known in which kinetic properties have been altered after modification of an enzyme and there are also cases in which new catalytic activities have been introduced [37,38].…”

Section: Internal Modificationsmentioning

confidence: 99%

Perspectives for biophysicochemical modifications of enzymes

Roig

Kennedy

1996

Journal of Biomaterials Science, Polymer Edition

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This article reviews the strategies and successes of modifying enzymes by means of biophysicochemical transformations. By judicious choice of methods, it has been possible to modify enzymes through physical interactions, chemical reactions and/or mutagenesis to alter a very wide range of properties ranging from stability and solubility on the one hand to catalytic activity and selectivity on the other.

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Kinetic analysis of regeneration by dilution of a covalently modified protein

Rakitzis

1990

Biochemical Journal

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An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.

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Kinetic analysis of protein modification reactions at equilibrium

Cited by 3 publications

References 28 publications

Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

Perspectives for biophysicochemical modifications of enzymes

Kinetic analysis of regeneration by dilution of a covalently modified protein

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