Mechanism-based enzyme inactivators, design

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. 

Since the initial description of a mechanism-based enzyme inactivator in the late 1960s, the field has been reviewed extensively from a variety of perspectives (Abeles and Maycock, 1976 Walsh, 1982, 1984 Rando, 1984 Silverman and Hoffman, 1984 Palfreyman et al., 1987 Silverman, 1988). Rather than to provide a comprehensive summary of the entire literature in this area, the goal of this chapter is to describe the classic approaches to inactivation of a variety of enzymes, drawing illustrations from the best understood examples. This review begins with a discussion of the criteria which define a mechanism-based enzyme inactivator and the strategies for design of such compounds. 

During the past three decades, besides the rational design of hundreds of molecules that have been synthesized and tested as suicide substrates. It also has come to light that nature Itself has known about this mechanistic mode of enzyme Inhibition and provided us with several extremely potent mechanism-based suicide Inactivators. Below are a few selected examples to demonstrate the mode of action of these Inhibitors. 

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). 

Mechanism-based irreversible inhibition occurs when a reactive metabolic intermediate is formed in situ that can (1) bind covalently with the prosthetic heme through N-alkyllary-lation (e.g., secobarbital), f"2 alkylate the apo-cytochrome (e.g., chloramphenicol or 2-ethy-nylnaphthalene), or (5) cause destruction of the prosthetic heme to products that irreversibly bind to the apocytochrome (e.g. CCI4) (158). These mechanism-based inactivators have primarily been designed and used for the selective inhibition of specific CYP enzymes and elucidating the mechanism of P450 reactions. Some drugs (e.g., aromatase inhibitors)... 

Several PLP-dependent enzymes catalyze elimination and replacement reactions at the y-carbon of substrates, an unusual process which provides novel routes for mechanism-based inactivation. An example of this class of enzymes is cystathionine y-synthase [0-succinylhomoserine (thiol)-lyase], which converts (7-succinyl-L-homoserine and L-cysteine to cystathionine and succinate as part of the bacterial methionine biosynthetic pathway (Walsh, 1979, p. 823). Formation of a PLP-stabilized o-carbanion intermediate activates the )8-hydrogen for abstraction, yielding j8-carbanion equivalents and allowing elimination of the y-substituent. The resulting j8,y-unsaturated intermediate serves as an electrophilic acceptor for the replacement nucleophile. Suitable manipulation of the j8-carbanion intermediate allows strategies for the design of inactivators which do not affect enzymes which abstract only the a-hydrogen. 

Fluorouracil is a mechanism-based inhibitor—it inactivates the enzyme by taking part in the normal catalytic mechanism. It is also called a suicide inhibitor because when the enzyme reacts with it, the enzyme commits suicide. The use of 5-fluorouracil illustrates the importance of knowing the mechanism for an enzyme-catalyzed reaction. If you know the mechanism, you may be able to design an inhibitor to turn the reaction off at a certain step. 

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