title: 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 acyl-group 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 (Ki= 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.
authors.affiliation: Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Adediran
authors.affiliation: College of Pharmacy, University of Michigan, Ann Arbor, MI 48105, USA.
authors.forename: Pan-Fen
authors.initials: PF
authors.lastname: Wang
authors.affiliation: Department of Chemistry, East Tennessee State University, Johnson City, TN, 37614, USA.
authors.forename: Abbas G
authors.initials: AG
authors.lastname: Shilabin
authors.affiliation: Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Baron
authors.affiliation: College of Pharmacy, University of Michigan, Ann Arbor, MI 48105, USA; Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
authors.affiliation: Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA. Electronic address: rpratt@wesleyan.edu.
authors.forename: R F
authors.initials: RF
authors.lastname: Pratt
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002254:Carbonates / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003460:Crystallization; D006867:Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000737:chemistry*; D007700:Kinetics; D008333:Mandelic Acids / Q000737:chemistry*; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000737:chemistry; D016958:Pseudomonas putida / Q000201:enzymology; D013379:Substrate Specificity; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001426:Bacterial Proteins; D002254:Carbonates; D006867:Hydrolases; D006877:Hydroxamic Acids; D008333:Mandelic Acids; D009596:Nitrophenols; D000099252:Fatty Acid Amide Hydrolases; C024719:4-nitrophenyl; C010413:mandelamide; C008642:4-nitrophenyl acetate; C019665:amidase; C000619257:mandelamide hydrolase, Pseudomonas putida
keywords: Amide signature enzymes; Inverse acyl-enzyme; Mandelamide hydrolase; Structure/activity; Substrates and inhibitors