Transition-state analogs of an aliphatic amidase

Abstract

The use of transition-state analogs in the elucidation of enzyme mechanisms has assumed importance in recent years [ 1,2] . The idea that synthetic compounds resembling the proposed transition-state should be extremely effective inhibitors compared to ground-state analogs, and should bind more effectively even than the substrate, has been substantiated. Recent studies with papain [3] and elastase [4] showing that aldehydes with the appropriate peptide side chains are extremely effective inhibitors has led to the proposal that the free aldehyde reacts with an amino acid residue (cysteine-papain; setie-elastase) in the active centre to form a hemithibacetal or hemiacetal; this being the ‘true’ transition-state analog on the reaction pathway to acylenzyme formation. Aldehydes, however, exist in appreciable amounts in aqueous solution as hydrates formed rapidly by a general base catalysed reaction [S] . At the present time there is no way of determining whether the true inhibitor is the free aldehyde or its hydrate. This paper presents evidence that, at least for the aliphatic amidase from Pseudomonas aeruginosa, it is the hydrated aldehyde that is the true inhibitor. T%is conclusion is substantiated by the observation that acetaldehyde-ammonia is a potent inhibitor and might be considered to be a transition-state analog for amide hydrolysis.