Specificity and mechanism of mandelamide hydrolase catalysis

Abstract

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an "inverse" acyl-enzyme where the small acylgroup occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (K i = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors.3