Enzymes high-affinity interaction

The structures of enzyme active sites, and other ligand binding pockets on enzymes, are ideally suited for high-affinity interactions with drug-like inhibitors. 

When ascorbate acts as an antioxidant or enzyme cofactor, it becomes oxidized to DHAA. Ascorbate and DHAA possess roughly equivalent bioavailability. Bioavailability is determined by the rates of absorption, distribution, and metabolism within the body, and by excretion. Ascorbate and DHAA are absorbed along the entire length of the human intestine (Malo and Wilson, 2000). For both the DHAA and ascorbate transport systems, initial rates of uptake saturate with increasing external substrate concentration, reflecting high-affinity interactions that can be described by Michaelis-Menten kinetics. 

In addition, the combination of addressing with the high-affinity interaction of avidin-biotin (association constant Ka = 10 M ) leads to an affinity-driven immobilization protocol for enzymes involving solely a single attachment point that fully retains its biological activity [32-35]. 

Chapter 5 also demonstrates that a combination of Lewis-acid catalysis and micellar catalysis can lead to accelerations of enzyme-like magnitudes. Most likely, these accelerations are a consequence of an efficient interaction between the Lewis-acid catalyst and the dienophile, both of which have a high affinity for the Stem region of the micelle. Hence, hydrophobic interactions and Lewis-acid catalysis act cooperatively. Unfortunately, the strength of the hydrophobic interaction, as offered by the Cu(DS)2 micellar system, was not sufficient for extension of Lewis-acid catalysis to monodentate dienophiles. 

Chemical modifications like alkylation with (A-ethylmaleimide (NEM) or oxidation with diamide that inhibit the phosphorylation activity of the enzyme did not seem to have any significant effect on the high affinity binding site when the enzyme was solubilized in the detergent decyl-PEG [69,41]. However, in the intact membrane these treatments reduced the affinity by a factor of 2-3. The reduction of the affinity was exclusively due to modification of the cysteine residue at position 384 in the B domain [69]. Apparently, the detergent effects the interaction between the B and C domains. 

It is worth noting here that inhibitors that interact with enzyme active site functionalities in ways that mimic the structure of covalent intermediates of catalysis can bind with very high affinity. This was seen in Chapter 1 with the example of statine-and hydroxyethylene-based inhibitors of aspartic proteases other examples of this inhibitor design strategy will be seen in subsequent chapters of this text. 

Figure 5.6 Biphasic concentration-response plot for an enzyme displaying a high- and low-affinity binding interaction with an inhibitor. In panel A, the data are fit to Equation (5.4) and the best fit suggests a Hill coefficient of about 0.46. In panel B, the data are fitted to an equation that accounts for two, nonidentical binding interactions Vj/v0 = (a/(l + ([/]/ICs0))) + ((1 - a)/(l+([t]/IC(o)))> where a is an amplitude term for the population with high binding affinity, reflected by IC , and IC 0 is the IC50 for the lower affinity interaction. (See Copeland, 2000, for further details.)...

Figure 5.7 Comparison of four-parameter fy-maxi mum, v-minimum. IC50, and h) and two-parameter (IC50 and h) fits of non-ideal concentration-response data. In panels A and B the data indicate a nonzero plateau at low inhibitor concentration that might reflect a low-amplitude, high-affinity second binding interaction. In panels C and D the data indicate a plateau at high inhibitor concentration that does not achieve full inhibition of the enzyme. There could be multiple causes of behavior such as that seen in panels C and D. One common cause is low compound solubility at the higher concentrations used to construct the concentration-response plot. Note that the discordance between the experimental data and the expected behavior is most immediately apparent in the plots that are fitted by the two-parameter equation.

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