Covalently binding enzyme inhibitors inactivation

Evaluation of the Mechanism of Inactivation of Covalently Binding Enzyme Inhibitors, 756... 

The first group of covalently binding enzyme inhibitors, the chemical modifiers, are small organic molecules, generally electrophiles, that are used to modify the enzyme s side chains in such a way as to produce a stable covalent bond. These are often used to study enzyme inactivation and to identify residues potentially involved in binding and catalysis. Some of the commonly used reagents are... 

The inherent complexity of the inactivation mechanisms of covalently binding enzyme inhibitors makes it necessary to evaluate their proposed modes of action carefully. An overview of the criteria for the study of irreversible inhibitors is provided below. 

This class of inhibitors usually acts irreversibly by permanently blocking the active site of an enzyme upon covalent bond formation with an amino acid residue. Very tight-binding, noncovalent inhibitors often also act in an irreversible fashion with half-Hves of the enzyme-inhibitor complex on the order of days or weeks. At these limits, distinction between covalent and noncovalent becomes functionally irrelevant. The mode of inactivation of this class of inhibitors can be divided into two phases the inhibitors first bind to the enzyme in a noncovalent fashion, and then undergo subsequent covalent bond formation. 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

Not all enzyme inhibitors bind through reversible interactions. In some cases enzymes are inactivated by formation of covalent complexes with inhibitory molecules. 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

Furans can also undergo covalent binding to macromolecules [20]. Furans, such as 8-methoxypsoralen, are epoxidized by Cyt P450 to yield a highly electrophilic species, which is so reactive that it covalently modifies an amino acid residue of CYP itself and thus inactivates the enzyme irreversibly (hence it is also in the class of suicide inhibitors) [21]. 

An irreversible inhibitor binds tightly, often covalently, to amino acid residues at the active site of the enzyme, permanently inactivating the enzyme. Examples of irreversible inhibitors are diisopropylfluorophosphate (DIPF), iodoacetamide and penicillin. 

Studies in recent years have revealed a number of remarkable drug interactions with irreversible or mechanism-based inhibitors of CYP3A, many of which can be attributed to inhibition of sequential intestinal and hepatic first-pass metabolism. Mechanism-based inhibition involves the metabolism of an inhibitor to a reactive metabolite, which either forms a slowly reversible metabolic-intermediate (MI) complex with the heme moiety or inactivates the enzyme irreversibly via covalent binding to the enzyme catalyzing the last step in the bioactivation sequence. As a result, mechanism-based inhibition is both... 

Inhibition of lipases, both by the substrate or the product, has been observed. In alcoholysis of methyl propanoate with n-propanol catalyzed by Candida antarctica lipase B (CALB), the alcohol was found to inhibit the enzyme resulting in a deadend complex [21]. Phosphate- and phosphonate-conlaining inhibitors are known to inhibit proteases. Studies of the inhibition of CALB have shown inhibition by diethyl p-nitrophenyl phosphate. The inactivation of the enzyme was caused by covalent binding of diethyl p-nitrophenyl phosphate in the active site [22]. 

L Irreversible inactivation. Inactivation by affinity labels leads to irreversible covalent bond formation between the enzyme and the inhibitor. Unlike the complex between and enzyme and a rapid, reversible inhibitor, the covalent enzyme-inhibitor complex is no longer in equilibrium with free enzyme and inhibitor. Therefore, exhaustive dialysis or gel filtration of the covalent enzyme-inhibitor complex cannot lead to the recovery of free, active enzyme. However, such experiments do not allow distinction among tight-binding, noncovalent inhibitors, affinity labels, and mechanism-based inactivators. 

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