Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function

Rakitzis, Emmanuel T.; Malliopoulou, Thalia B.

doi:10.1042/bj2370589

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Biochemical Journal

1986

DOI: 10.1042/bj2370589

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Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function

Emmanuel T. Rakitzis¹,

Thalia B. Malliopoulou²

Abstract: A mathematical treatment of modification-induced enzyme protein inactivation is presented, and it is shown that, at initial reaction conditions, the ratio of the first derivative of the equation describing enzyme activity loss to the first derivative of the equation describing protein groups modification is equal to the fractional concentration of enzyme protein reactive groups, or group reactivities, essential for catalytic function.

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Cited by 5 publications

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Phenylglyoxal modification of arginines in mammalian D‐amino‐acid oxidase

Vanoni¹,

Simonetta²,

Curti³

et al. 1987

European Journal of Biochemistry

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The presence of arginine in the active center of D-amino-acid oxidase is well documented although its role has been differently interpreted as being part of the substrate-binding site or the positively charged residue near the N'-C2 = 0 locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in Damino-acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine-directed reagent. Loss of catalytic activity followed pseudo-first-order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8-mercapto-FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme-binding ability. Analyses of time-dependent activity loss versus arginine modification or [ ''C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active-site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8-mercapto derivative of D-amino-acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N-terminal portion of the polypeptide chain and point to a role of phenylglyoxalmodifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme.D-Amino-acid oxidase has been widely studied with respect to its kinetic and structural properties. Some amino acid residues involved in catalysis, such as tyrosine, lysine and histidine, have been identified by chemical modification experiments and localized within the enzyme primary structure [l -41. The work of several groups [5-71 indicated the involvement of arginine residues in the D-amino-acid oxidase active site and pointed out the role of an arginine in the binding site of the carboxylate moiety of the substrate. Evidence was also given that one of the 1,2-cyclohexanedionemodifiable arginines of D-amino-acid oxidase is the postulated positively charged group which interacts with the FAD N'-C2 = 0 locus [8]. With the aim of identifying the D-amino-acid oxidase active-site arginine we first undertook the analysis of enzymic digests of 1,2-cyclohexanedione-modified enzyme. However, our attempts were unsuccessful owing to the instability of the cyclohexanedione-arginine adduct and the low sensitivity and reliability of its detection method [9]. Phenylglyoxal was then used throughout this work since it gives a more stable derivative and it is readily available in the radioactive form [lo, 111. Analyses of modification of apo and holo forms of D-amino-acid oxidase strongly support the inv...

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Phenylglyoxal modification of arginines in mammalian D‐amino‐acid oxidase

Vanoni¹,

Simonetta²,

Curti³

et al. 1987

European Journal of Biochemistry

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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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Kinetics of Protein Modification Reactions: Analysis of Modification-Induced Protein Unfolding

Rakitzis

1987

Journal of Enzyme Inhibition

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A mathematical treatment of a two-sited, modification-induced protein unfolding model is presented, and it is shown that the dependence of the concentration of modified protein groups on reaction time is described by a linear, second-order, differential equation with nonzero right hand side. The analytic solution of this equation consists of a summation of exponential functions of reaction time. By assigning arbitrary values to the modification and isomerization rate constants of these equations, simulated cases of protein modification are presented, and the apparent end-point of the reaction is determined graphically. It is found that the apparent end-point of the reaction is, in most cases studied, different from the true value of two groups modified per protein molecule, and is a function of both the modification, and isomerization rate constants of the model. The first derivative of the protein modification reaction, at the start of the reaction, [E]'mod (0), is determined, for the same simulated cases of protein modification, by two different analytical methods. It is found that the [E]'mod(0) value, obtained from graphical and numerical analysis data, is in most cases in good agreement with the value expected from first principles. Finally, the different irreversible enzyme inhibition forms, contingent upon the different kinds of the enzyme inactivation-protein modification relationships of the protein modification model under consideration, are presented and discussed.

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Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function

Cited by 5 publications

References 15 publications

Phenylglyoxal modification of arginines in mammalian D‐amino‐acid oxidase

Phenylglyoxal modification of arginines in mammalian D‐amino‐acid oxidase

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

Kinetics of Protein Modification Reactions: Analysis of Modification-Induced Protein Unfolding

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