Kinetic analysis of regeneration by dilution of a covalently modified protein

Abstract

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.

Perspectives for biophysicochemical modifications of enzymes

“…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].…”

Transient Inactivation of Almond Mandelonitrile Lyase by 3-Methyleneoxindole: A Photooxidation Product of the Natural Plant Hormone Indole-3-Acetic Acid

“…Briefly, samples of MNL were inactivated by 0.2 mM MOI and then diluted 10, 15, 25, 50, and 100-fold with citrate buffer, pH 5.5. The time course for recovery of MNL activity was monitored, and the data from each dilution were plotted as In [(E^ -EJ/ÇE,^ -E^] vs time 25 , where E 0 is the enzyme activity at the start of the dilution-induced reactivation event, Έ^ is the activity at reaction time t, and Egq is the activity at equilibrium (Figure 3.7).…”

“…The slopes of the five lines in Figure 3.7 were then used to determine &_ 2 according to equation 3.Ó 25 :…”

“…As Rakitzis points out 25 , the occurrence of reaction situations more complicated than that described in equation 3.5 (such as presence of two kinds of modifiable groups on the protein, or conformational isomerism) is probably not amenable to interpretation by the kinetic analysis described above. In our case, these reaction situations were ruled out, since the value we obtained for &_ 2 was a^s0 confirmed by an independent experimental method (section 3.3.11).…”

Utilization of the free energy of the reversible binding of protein and modifying agent towards the rate-enhancement of protein covalent modification

“…Protein modification reactions may exhibit an enhancement of rate when compared with the reaction of the same modifying agent with a micromolecular model compound possessing the same modifiable group as the protein under study. The question of the maximum extent of rate-enhancement of protein modification may be answered by the acceptance of certain formalistic assumptions, namely the description of the protein modification reaction by a single exponential function of reaction time, and also of an acceptable rate of dilution-induced enzyme re-activation (Rakitzis, 1989(Rakitzis, , 1990. In the present paper it is shown that the maximunj extent of affinity-mediated rateenhancement of protein modification may be determined if the entropy of activation of the reaction of the modifying agent under study with a model micromolecular compound is known.…”