Inhibitor binding others

A noncompetitive inhibitor is one that inhibits the enzyme and its inhibitory activity is unaffected by substrate, i.e., it will inhibit the enzyme to the same degree whether the substrate is present or not. This is generally thought to occur by the inhibitor binding at some site other than the substrate-binding site but in a way that inactivates the enzyme, e.g., induced conformational change of the active site. Therefore, we may have inhibitor binding reversibly to free enzyme [Eq. (3.22)] or to the enzyme substrate complex [Eq. (3.23)], but in both cases the bound enzyme is inactive. 

As the investigation of the interactions between H DAC inhibitors and the enzymes are an important issue, competition assay systems are helpful implements in facilitating the characterization of inhibitor binding. Such a competition binding assay that has been developed for histone deacetylases is based on fluorescence resonance energy transfer (FRET) between tryptophan residues of the histone deacetylase and a fluorescent HDAC inhibitor [38]. In competition with other... 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

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