RT Journal Article SR Electronic T1 Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.110.065458 DO 10.1124/mol.110.065458 A1 Anthony L Albiston A1 Vi Pham A1 Siying Ye A1 Leelee Ng A1 Rebecca A Lew A1 Phillip E Thompson A1 Jessica K Holien A1 Craig J Morton A1 Michael W Parker A1 Siew Yeen Chai YR 2010 UL http://molpharm.aspetjournals.org/content/early/2010/07/13/mol.110.065458.abstract AB Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orientate with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast the two 4-quinolinyl derivatives orientate in a different manner interacting with the Zn2+ ion via the quinoline nitrogen and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that is substrate dependent. Phe544 is also important for inhibitor binding as these mutations altered the Ki in some cases and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.