Mechanism-based enzyme inactivation acids

Mechanism-based enzyme inactivators are also powerful tools in the determination of enzyme mechanisms. Because some understanding of the enzyme mechanism is required for the design of an inactivator, the success of the compound provides support for the mechanistic hypothesis. Analysis of the intermediates and products of an inactivation reaction can be extremely useful in illuminating the normal mechanism of enzymic catalysis. For example, the covalent modification of an enzyme by a mechanism-based inactivator facilitates isolation of active site peptides and identification of cataiytically relevant amino acids. 

The second criterion for mechanism-based enzyme inactivation concerns the stoichiometry of enzyme modification. If mechanism-based inactivation of an enzyme is the result of specific covalent modification of an essential active site amino acid residue, then one radiolabeled inactivator molecule should be incorporated per active site. The stoichiometry of specific radiolabeling should also correlate with the extent of inactivation. In multimeric enzymes which display negative cooperativity, it is possible to observe complete inactivation following substoichiometric modification of the enzyme (Johnston et al., 1979). 

A significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Mechanistic investigations have shown that these compounds behave as suicide inhibitors (preferably called mechanism-based inactivators) in the sense that they are recognized by /3-lactamases as substrates, but the great stability of the acyl-enzyme intermediate blocks turnover of the enzyme [46] [47]. /3-Lactamase inhibitors can be divided into two classes, class I and class II class-I inhibitors (e.g., clavulanic acid (5.12)), in contrast to those of class II (e.g., olivanic acid (5.15)), have a heteroatom at position 1 that can lead to ring opening at C(5). The mechanistic consequences of this difference in structure are illustrated by the general scheme in Fig. 5.3. 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

The substrates used to generate the enamines to date include both benzylidenepyruvic acids 10 and benzoylformic acids 4. In 1983 it was reported that ( )-4-/7-chlorophenyl-2-oxo-3-butenoic acid (CPB, 20, X = p-C 1) acts as a mechanism-based inactivator of PDC60, and on decarboxylation it generates a new absorbance on the enzyme with 2max near 440 nm61. 

Clavulanic acid is also a mechanism-based inhibitor and its mode of action is believed to involve ring opening of the initially formed acyl-enzyme complex (18) to the keto-derivative (19), which may then tautomerise to the hydrolytically more stable -amino-acrylate (20) Scheme 6.4). This transiently inhibited form may hydrolyse to re-release active enzyme or react further with the enzyme to produce irreversibly inhibited forms. It has been shown that approximately 115 molecules of clavulanic acid are destroyed per molecule of enzyme before the j8-lactamase is irreversibly inactivated. Whilst irreversibly inactivated forms are known to exist, the nature of these products is not yet known. Possible structures are (21) and... 

The antibiotic chloramphenicol is oxidized by CYP monooxygenase to chloramphenicol oxamyl chloride formed by the oxidation of the dichloromethyl moiety of chloramphenicol followed by elimination of hydrochloric acid " (Figure 33.6). The reactive metabolite reacts with the e-amino group of a lysine residue in CYP and inhibits the enzymatic reaction progressively with time. This type of inhibition is a time-dependent inhibition or a mechanism-based inhibition or inactivation, and the substrate involved historically has been called a suicide substrate because the enzymatic reaction yields a reactive metabolite, which destroys the enzyme. ... 

After the initial discovery that 0-fluoromethylene-substituted amines (e.g., 184, Table 1) were potent, mechanism-based inhibitors of monoamine oxidase (MAO) (41), the concept was successfully broadened to include most of the common amine oxidases (Table 1). This approach was also used to design inhibitors of y-aminobutyric acid transaminase both the a- and 0- substituted amino acids 189 and 190 were found to inactivate this enzyme. Recently, applica-tion of this concept to the design of inhibitors of S-adenosyl-homocysteine hydrolase (SAH) has led to the discovery of very potent inhibitors of this enzyme (e.g., 176, Table 1). 

A mechanism-based inhibitor may be defined as a chemically unreactive compound that is treated by the target enzyme as a substrate, but instead of forming the usual product, it is converted into a highly reactive species via the normal catalytic mechanism. Prior to release from the active site, the reactive intermediate may alkylate amino acid functional groups, forming a new covalent bond and inactivating the enzyme (90). Irreversible, mechanism-based inactivation is typified by first-order, time-dependent loss of enzyme activity saturation kinetics inactivation protection by substrates and reversible inhibitors failure to recover activity following dialysis and usually a chemical stoichiometry of one covalent adduct formed per enzyme active site. 

A second new class of MAO mechanism-based inactivators, (aminoalkyl)tri-methylsilanes, have been reported by Silverman and Banik (114). The idea for this class of MAO inactivators is based on the known activation of the carbon-silicon bond toward homolytic cleavage reaction when the silicon atom is /3 to a radical cation (115, 116). The aminomethyl-, aminoethyl-, and (amino-propyl)trimethylsilanes are all pseudo-first-order time-dependent inactivators of beef liver MAO that reduce the flavin cofactor during the inactivation reaction. Since denaturation of the inactivated enzyme allows flavin leoxidation, covalent bond formation might be to an amino acid residue (114). The stabilities of the enzyme adducts from the (aminoalkyl)trimethylsilanes were found to be differ-... 

The pyridoxal phosphate-dependent enzymes have been a major focal point in the development of mechanism-based inactivators. Pyridoxal phosphate (PLP) is utilized in resonance stabilization of carbanions at the a- and /3-carbons of amino acids in a variety of reactions which lead to chemical transformations at the a-, /3-, and y-carbons of the substrate (Walsh, 1979). These carbanion equivalents... 

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