Specificity and mechanism of mandelamide hydrolase catalysis

S A Adediran; Pan-Fen Wang; Abbas G Shilabin; Charles A Baron; Michael J McLeish; R F Pratt

doi:10.1016/j.abb.2017.01.010

Specificity and mechanism of mandelamide hydrolase catalysis

Arch Biochem Biophys. 2017 Mar 15:618:23-31. doi: 10.1016/j.abb.2017.01.010. Epub 2017 Jan 24.

Authors

S A Adediran¹, Pan-Fen Wang², Abbas G Shilabin³, Charles A Baron¹, Michael J McLeish⁴, R F Pratt⁵

Affiliations

¹ Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
² College of Pharmacy, University of Michigan, Ann Arbor, MI 48105, USA.
³ Department of Chemistry, East Tennessee State University, Johnson City, TN, 37614, USA.
⁴ 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.
⁵ Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA. Electronic address: rpratt@wesleyan.edu.

PMID: 28129982
DOI: 10.1016/j.abb.2017.01.010

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 (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.

Keywords: Amide signature enzymes; Inverse acyl-enzyme; Mandelamide hydrolase; Structure/activity; Substrates and inhibitors.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Amidohydrolases / chemistry*
Bacterial Proteins / chemistry*
Binding Sites
Carbonates / chemistry
Catalysis
Catalytic Domain
Crystallization
Hydrolases / chemistry*
Hydrolysis
Hydroxamic Acids / chemistry*
Kinetics
Mandelic Acids / chemistry*
Molecular Conformation
Mutagenesis, Site-Directed
Nitrophenols / chemistry
Pseudomonas putida / enzymology
Substrate Specificity

Substances

Bacterial Proteins
Carbonates
Hydroxamic Acids
Mandelic Acids
Nitrophenols
4-nitrophenyl
mandelamide
4-nitrophenyl acetate
Hydrolases
Amidohydrolases
fatty-acid amide hydrolase
amidase
mandelamide hydrolase, Pseudomonas putida