Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor

• Published in: Biochimica et biophysica acta 2011-05, Vol.1814 (5), p.638-646
• Main Authors: Ernberg, Karin, Zhong, Bo, Ko, Kristin, Miller, Larry, Nguyen, Yen Hoang le, Sayre, Lawrence M., Guss, J. Mitchell, Lee, Irene
• Format: Article
• Language: English
• Subjects: Allenic amine; Amine Oxidase (Copper-Containing) - antagonists & inhibitors; Amines - chemistry; Amines - pharmacology; Arthrobacter - enzymology; Arthrobacter globiformis; Arthrobacter globiformis amine oxidase; Coenzymes - chemistry; Crystal structure; Dihydroxyphenylalanine - analogs & derivatives; Dihydroxyphenylalanine - chemistry; Inhibition potency; Mechanism-based inactivator; Models, Molecular; Molecular Structure
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2010.12.016

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold Km). Comparing the IC50 values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site. [Display omitted] ► 5-phenoxy-2,3-pentadienylamine inactivates Arthrobacter globiformis amine oxidase (AGAO). ► 6-phenyl-2,3-hexadienylamine inactivates AGAO. ► Crystal structures of AGAO attached to inactivators reveal mechanism of action. ► IC50 values reveal inhibition potency and suggest selectivity in AGAO inactivation. ►Structural framework to guide the design of selective mechanism-based inhibitors.