Covalent bonds enzyme-inhibitor

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... 

Covalent bond (Section 1 3) Chemical bond between two atoms that results from their shanng of two electrons COX 2 (Section 26 6) Cyclooxygenase 2 an enzyme that cat alyzes the biosynthesis of prostaglandins COX 2 inhibitors reduce pain and inflammation by blocking the activity of this enzyme... 

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 efficiency of inactivation by covalent bond formation vs release of the reactive species into solution has been described by its partition ratio. The most efficient inactivators have catalytic partition ratios of 0, in which case each inhibitor molecule leads to inactivation of the enzyme. To this date, many of these inhibitors have been designed, and alternative names like suicide substrate, Trojan Horse inactivator, enzyme induced inactivator, inhibitor, and latent inactivator have been used for this class of inhibitors. A number of comprehensive reviews are available (26—32). 

Affinity Labels. Active site-directed, irreversible inhibitors or affinity labels are usually substrate analogues that contain a reactive electrophilic functional group. In the first step, they bind to the active site of the target enzyme in a reversible fashion. Subsequentiy, an active site nucleophile in close proximity reacts with the electrophilic group on the substrate to form a covalent bond between the enzyme and the inhibitor, typically via S 2 alkylation or acylation. Affinity labels do not require activation by the catalysis of the enzyme, as in the case of a mechanism-based inhibitor. 

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). 

The enzyme catalyzes the hydrolysis of an amide bond linkage with water via a covalent enzyme-inhibitor adduct. Benzoxazinones such as 2-ethoxy-4H-3,l-benzoxazin-4-one [41470-88-6] (23) have been shown to completely inactivate the enzyme in a competitive and stoichiometric fashion (Eigure 5). The intermediate (25) is relatively stable compared to the enzyme-substrate adduct due to the electron-donating properties of the ortho substituents. The complex (25) has a half-life of reactivation of 11 hours. 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Neuraminidases are enzymes present in viruses, bacteria, and parasites. They are implicated in serious diseases such as cholera, meningitis and pneumonia. Neuraminidase from influenza virus aids the transmission of the virus between cells and maintains viral infectivity. In different strains of influenza several amino acids are conserved, especially in the active site, giving rise to hopes of finding a single inhibitor (and so a drug) for all the neuraminidase enzymes from influenza strains. The crucial question is whether a covalent bond is formed between the enzyme and the reaction intermediate. 

The calculations found there was no covalent intermediate in the viral neuraminidase reaction and the intermediate was more likely to be hydroxylated directly. Because there is only a small energy difference between the two options (formation of a covalent bond or direct hydroxylation) Thomas et al. proposed it might be possible to design inhibitors covalently bound to the enzyme. 

Suicide substrates and quiescent affinity labels, unlike the other types of inhibitors discussed in this chapter, form covalent bonds with active site nucleophiles and thereby irreversibly inactivate their target enzymes. A suicide substrate,191 also described by Silverman in a comprehensive review1101 as a mechanism-based inactivator, is a molecule that resembles its target enzyme s true substrate but contains a latent (relatively unreactive) electrophile. When the target enzyme attempts to turn over the... 

The closest organic specie to the inorganic boric acid are the boronic acids generally described as R-B(OH)2. Boronic acids have been shown to act as inhibitors of the subtilisins. X-ray crystallographic studies of phenylboronic acid and phenyl-ethyl-boronic acid adducts with Subtilisin Novo have shown that they contain a covalent bond between the oxygen atom of the catalytic serine of the enzyme and the inhibitor boron atom (Matthews et al, 1975 and Lindquist Terry, 1974). The boron atom is co-ordinated tetrahedrally in the enzyme inhibitor complex. It is likely that boric acid itself interacts with the active site of the subtilisins in the same manner. 

Some of these inhibitors are reversible since they can easily be removed from the binding site, so the enzyme regains its activity. But in some instances a covalent bond is formed between the enzyme and the inhibitor, in which case the catalytic activity is not restored when the enzyme is separated from the solution containing the inhibitor. Such inhibitors are called irreversible. 

Such drugs interact very rapidly and reversibly with the enzyme. Once bound, a time-dependent reaction of an enzyme nucleophile with bound inhibitor occurs, resulting in formation of a covalent bond. Although... 

All the commonly used MAOIs (monoamine oxidase inhibitors), exemplified by phenelzine, isocarboxazid and pargyline, are irreversible inhibitors of both forms of the enzyme, forming covalent bonds with the active sites... 

If the bond formed between an enzyme and an inhibitor is a strong covalent bond and... 

The site of action in the 3-lactam antibiotics is muramoylpentapeptide carboxypeptidase, an enzyme that is essential for cross-linking of bacterial cell walls. The antibiotic resembles the substrate of this enzyme (a peptide with the C-terminal sequence D-Ala-D-Ala) and is therefore reversibly bound in the active center. This brings the 3-lactam ring into proximity with an essential serine residue of the enzyme. Nucleophilic substitution then results in the formation of a stable covalent bond between the enzyme and the inhibitor, blocking the active center (see p. 96). In dividing bacteria, the loss of activity of the enzyme leads to the formation of unstable cell walls and eventually death. 

These drugs, which form stable complexes with MAO and thereby inhibit its action have long been used in medicine as antidepressants, and are referred to as MAO inhibitors. It is possible, that MAO inhibitors act not by complexation with the enzyme, but by forming covalent bonds that is by irreversibly inactivating the enzyme. 

Early classical MAO inhibitors used clinically, such as tranylcypromine and phenelzine, generate reactive species, which are trapped by MAO by formation of strong covalent bonds with the enzyme [32,64,85]. For this reason, effects could be of... 

Di- and trifluoromethyl ketones inhibit a great number of esterases and proteases with often very high inhibition constants (cf. Chapter 7). Although the fluorinated ketone is covalently bonded to the nucleophilic residue of the enzyme, the inhibition is reversible, as the inhibitor could be displaced by another nucleophile. The covalent nature of the interactions as well as the tetrahedral structure of the adducts have been demonstrated by kinetic studies, by NMR experiments, and by the X-ray diffraction of the enzyme-substrate complexes. ... 

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