Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications

Carefully designed molecules that are intimately related to the reaction mechanism of enzymes are often highly selective and potent inhibitors that serve as extremely useful chemical probes for understanding the reaction mechanism and structure of enzymes. This article describes the design, synthesis, and applications of specific inhibitors of two mechanistically distinct groups of enzymes, ATP‐dependent amide ligases and Ser‐ and Thr‐hydrolases. Our strategy is based on the premise that stable analogues of the transition state (transition‐state analogues) are highly potent inhibitors that serve as good mechanistic probes, and that a key structure of a good inhibitor of one enzyme is also utilized for the inhibitors of other enzymes that share the same chemistry in their catalyzed reactions, irrespective of the degree of structural similarity and evolutionary link between the enzymes. According to these principles, we designed and synthesized a series of phosphinate‐ and sulfoximine‐based transition‐state analogue inhibitors of glutathione synthetase, γ‐glutamylcysteine synthetase and asparagine synthetase. For the second group of enzymes, we synthesized a γ‐monofluorophosphono glutamate analogue for mechanism‐based affinity labeling of γ‐glutamyltranspeptidase and fluorescent phosphonic acid esters for the active‐site titration of lipase. These inhibitors were used successfully as ligands for detailed kinetic analyses, X‐ray crystallography, and mass analysis of the enzymes to identify the key amino acid residues responsible for catalysis and substrate recognition in the transition state. © 2005 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 5: 209–228; 2005: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20045 Carefully designed transition‐state analogues are highly useful chemical probes for understanding the reaction mechanism and structure of enzymes. We present the rational design and synthesis of a series of phosphinate‐ and sulfoximine‐based transition‐state analogues as inhibitors of ATP‐dependent ligases. For a mechanism‐based affinity labeling agent of γ‐glutamyl transpeptidase and lipase, a γ‐monofluorophosphono glutamate analogue and a fluorescent phosphonic acid ester were synthesized, respectively. These inhibitors served as excellent ligands for detailed kinetic analyses, X‐ray crystallography, and mass analysis of the enzymes to identify the key amino acid residues responsible for