Kinetics of irreversible enzyme inhibition: The interpretation of the fractional enzyme activity vs. extent of protein modification plot

Rakitzis, Emmanuel T.

doi:10.1016/0022-5193(80)90328-8

Cited by 11 publications

(9 citation statements)

References 21 publications

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“…The second isoenzyme possesses three reactive residues per molecule of protein and is modified with a rate constant of 0.1 min-. [Both cases shown in the Figure have been presented in Rakitzis (1980b). ] combine statistical and time-dependence considerations when studying the modification of a catalytically active protein.…”

Section: Ligand Concentrationmentioning

confidence: 70%

“…Such plots have been used to obtain, by a process of extrapolation, the maximum extent of protein modification, i.e., the total number of reactive groups per protein molecule. It is common practice to construct and interpret such plots of fractional enzyme activity versus extent of protein modification on an intuitive basis, despite the firm theoretical treatment of this topic by Tsou (1962), and the elaborations on this by Horiike & McCormick (1979), Stevens & Colman (1980), and Rakitzis (1978aRakitzis ( , 1980b. Tsou (1962) has employed a statistical method of studying the relationship between fractional enzyme activity and extent of protein modification.…”

Section: Ligand Concentrationmentioning

confidence: 99%

“…Rakitzis (1978a) has pointed out that, in the case of enzyme protein modification reactions which are described by summations of exponential functions of reaction time, the conclusions of Tsou (1962) can also be arrived at by a juxtaposition of the equation describing protein modification and the equation describing enzyme inactivation. It is accordingly seen that it is wrong to extrapolate the initial portion of the plot of fractional enzyme activity versus number of groups modified per molecule of protein, and interpret the intercept of this on the axis representing the number of groups modified to mean the number of groups of the 'fast' reacting set; an exception to this is strong irreversible binding co-operativity (Rakitzis, 1980b). This fundamental mistake has also been pointed out by Horiike & McCormick (1979, and by Stevens & Colman (1980).…”

Section: Ligand Concentrationmentioning

confidence: 99%

“…This fundamental mistake has also been pointed out by Horiike & McCormick (1979, and by Stevens & Colman (1980). A large number of authors have used the plot of fractional enzyme activity versus extent of protein modification to distinguish between 'fast' and 'slow' sets of residues, without a proper analysis of this plot (e.g., Di Pietro et al, 1979;Petz et al, 1979;Bond et al, 1980;de Kok et al, 1980;Huber et al, 1982;Makinen et al, 1982;Inano & Tamaoki, 1983, as well as the papers cited by Rakitzis, 1980b). Hypothetical cases of enzyme protein modification which may lead to an erroneous interpretation of Tsou plots are shown in Fig.…”

Section: Ligand Concentrationmentioning

confidence: 99%

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Kinetics of protein modification reactions

Rakitzis¹

1984

Biochemical Journal

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Section: Ligand Concentrationmentioning

confidence: 70%

Section: Ligand Concentrationmentioning

confidence: 99%

Section: Ligand Concentrationmentioning

confidence: 99%

Section: Ligand Concentrationmentioning

confidence: 99%

See 2 more Smart Citations

Kinetics of protein modification reactions

Rakitzis¹

1984

Biochemical Journal

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“…This plot can give misleading results when used to try to establish the number of 'fast' and 'slow' reacting and 'essential' residues in the case of a complex multistep inactivation. However, the maximum number of modifiable rcsidues is correctly assigned by extrapolation of the curve fitting the experimental data to the abscissa axis, regardless of the reaction scheme used to describe the process (Rakitzis, 1978(Rakitzis, , 1980. The pattern of arginine modification is biphasic and paralleled the time course of enzyme inactivation (Fig.…”

Section: Characterization Of the Amino Acid Residues Modified By Phenmentioning

confidence: 99%

Chemical modification of functional arginyl residues in beef kidney d‐Aspartate oxidase

Tedeschi¹,

Negri²,

Ceciliani³

et al. 1992

European Journal of Biochemistry

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Chemical modification of beef kidney u-aspartate oxidase by phenylglyoxal is a biphasic process involving the transient formation of an enzymatic species with a decreased activity versus dicarboxylic substrates, an increased activity versus D-proline and a new activity versus other monocarboxylic D-amino acids which is absent in the native protein. Prolonged incubation with the modifier causes complete inactivation of the enzyme. The presence of the competitive inhibitor L-tartrate in the incubation mixture prevents enzyme inactivation. Kinetic and structural data suggest that complete loss of activity is paralleled by modification of eight arginine residues, of which two are critical for the specificity and the activity of the enzyme. We propose that the two essential arginine residues are located in the substrate binding site of D-aspartate oxidase.D-Aspartate oxidase is a flavoprotein which catalyzes the oxidative deainination of dicarboxylic D-amino acids by O2 with the production of H,02, NH3 and the corresponding 2-oxoacids. Reccntly the enzyme from beef kidney cortex has been purified to homogeneity and characterized both for its spectrophotometric properties and kinetic mechanism (Negri et al., 1987(Negri et al., , 1988. These studies showed that u-aspartate oxidase shares many features with u-amino acid oxidase, which is considered to be a model enzyme for the oxidasel dehydrogenase class of flavoproteins (Massey and Hemmerich, 1980). In particular, the kinetic mechanism of the two enzymes is very similar, suggesting a common design for their active site. Substrate and inhibitor specificity, however, is completely different, since D-aspartate oxidase binds dicarboxylic molecules while u-amino acid oxidase is specific for monocarboxylic ones (Hamilton, 1985). This observation suggests the presence in the active site of D-aspartate oxidase of an additional basic residue with respect to D-amino acid oxidase which confers its specificity for dicarboxylic ligands.Indication for a possible role in catalysis of arginine residues in waspartate oxidase was obtained by preliminary studies on partially purified enzyme conducted using both phenylglyoxal and 2,3-butanedione (Crifi, et al., 1977). The availability of a well characterized homogeneous enzyme has prompted us to reinvestigate the effect of chemical modification of waspartate oxidase by phenylglyoxal with respect to both the inhibition in catalytic activity and the spectral propertics of the enzyme. Phenylglyoxal has been widely used to map the active site of several flavoproteins, including D-amino acid oxidase (Vanoni et al., 1987), ferredoxin-NADP' reductase (Zanetti et al., 1979; Sancho et al., 1990), lactate oxidase (Peters et al., 1981), p-hydroxybenzoate hydroxylase (Shoun et al., 1980) and flavocytochrome b2 (Ghrir and Lederer, 1984).The data reported in this study suggest that two arginine residues are involved in the binding of the substrate to D-aspartate oxidase. EXPERIMENTAL PROCEDURES MaterialsD-Aspartate oxidase was purified from beef kid...

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Effects of chemical modification on enzymatic activities and stabilities

Sadana

Henley

1986

Biotech & Bioengineering

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The influence of chemical modification on the initial specific activity, residual activity, and deactivation kinetics of various enzymes is analyzed using a series mechanism. This straightforward multistate sequential model presented is consistent with the enzyme deactivation data obtained from different fields. The enzymes are placed in five different categories depending on the effect of chemical modification on initial specific activity and residual activity or stability. Wherever possible, structure-function relationships are described for the enzymes in the different categories. The categorization provides one avenue that leads to further physical insights into enzyme deactivation processes and into the enzyme structure itself.

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Kinetics of irreversible enzyme inhibition: The interpretation of the fractional enzyme activity vs. extent of protein modification plot

Cited by 11 publications

References 21 publications

Kinetics of protein modification reactions

Kinetics of protein modification reactions

Chemical modification of functional arginyl residues in beef kidney d‐Aspartate oxidase

Effects of chemical modification on enzymatic activities and stabilities

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