Kinetic analysis of the influence of inverse effectors (inhibitors and activators) on enzymatic (transport) activity of proteins
The method is proposed for calculation of the most important parameters of the two-stage enzymatic or transport process – modification factors ? and ? (which characterize the effector action mechanism) as well as the inhibition constant Kі or activation constant Ka (characterize the effector affinity for protein). The method was derived as based on the analysis of kinetic regularities of the action of reversible effectors (inhibitors and activators) on the catalytic (transport) activity of proteins. The method is based on the titration of enzymatic (transport) protein by the substrate with the absence and with presence of the effector taken in one of concentrations as well as determination (under the fixed substrate concentration) of the inhibition coefficient i0.5 (in case of the inhibitor action) or the activation coefficient а0.5 (in case of the activator action).
Practical use of the method has been demonstrated on the example of reversible inhibition to eosine Y (2?,4?,5?,7?-tetrabromofluorescein) of the reaction of enzymatic hydrolysis of ATP catalyzed by highly purified transport Ca2+, Mg2+-ATPase isolated from the smooth-muscle sarcolemma. In this case the inhibitory effect is characterized by the following parameters: ? = 6–8 more than 1; ? = 0.50–0.53 less than 1; inhibition constant Kі = 10-9–10-8 M. Consequently, judging from the values of ? and ?, the eosine Y effect on the analyzed Ca2+, Mg2+-dependent ATP-hydrolase enzymatic reaction is based on the mechanism of the mixed inhibition (one can observe the inhibition of the both stages of enzymatic transformation – the substrate binding with the enzyme and decomposition of “Michaelis complex” in the direction of formation of the reaction products). The inhibitor itself, in correspondence with Ki values is characterized by rather high affinity for Ca2+, Mg2+-ATPase. It is supposed that the proposed approach can be useful when identifying the type of the reversible effector action on the enzymatic (transport) activity of proteins, estimation of real affinity of the inhibitors and activators for the latter.
Published at the site: 2005-02-02
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