Enzyme/enzymatic suicide substrates

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. ... 

Figure 13.2. Rescue assay. The initial straight tine shows product formation by enzyme in the absence of inhibitor. An exponential decrease in rate follows addition of the suicide substrate. Upon addition of the nucleophile at time t, which consumes all excess inhibitor, a partial recovay of enz3une activity is observed. The final enzymatic rate is dependent on [1] and t.

Initiated by a paper from Carrell and co-workers [52] reporting the structure of D-xylose isomerase from Streptomyces rubiginosus refined to 1.9 A resolution, in which the authors claimed experimental support for the enediol mechanism, controversy on the true mechanism of the enzymatic catalysis arose. This group had designed the aldose analogue 32, a suicide inhibitor of the isomerase, and had determined three crystal structures that of the native enzyme, of the enzyme with bound substrate/product, and the enzyme after reaction with the mechanism-based inhibitor (Scheme 10). 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

Five novel carbonates/ designed as suicide (mechanism-based) inhibitors of acetylcholinesterase, were synthesized and evaluated against the enzyme in vitro and screened for insecticidal activity. The design strategy of inhibition was based on the isosteric relationship of carbonates to the ester of the natural substrate acetylcholine, and on the release of electrophilic quinone methides or alpha-chloroketones at the active site after enzymatic carbonate hydrolysis. Most coirpounds were inhibitory in vitro, with good specificity for acetylcholinesterase. Some showed modest insecticidal activity. Results of kinetic studies on one analog were consistent with mechanism-based inhibition. 

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