MALDI-TOF MS/MS analysis of the hNAAA tryptic peptide T10-β after covalent modification.

<p>Tandem MALDI-TOF MS/MS spectra of the T10-β peptide (sequence: CTSIVAQDSR) demonstrates covalent modification of Cys126 by both AM6701 (Panel (A)) and <i>N-</i>Cbz-serine β-lactone (Panel (B)).</p

Representation of the active site of hNAAA after treatment with <i>N-</i>Cbz-serine β-lactone.

<p>Homology model illustrates acylated catalytic nucleophile Cys126 after treatment with <i>N-</i>Cbz-serine β-lactone.</p

Potencies of hNAAA inhibitors.

<p>The <i>k</i>_inact and <i>K</i>_I values for the covalent inhibitors were obtained as described in the Experimental Procedures. The IC₅₀ values were calculated after 2 hours preincubation of the enzyme and inhibitor before addition of the substrate. Values are averages ± SD of three independent experiments.</p

Representation of the active site of hNAAA after treatment with AM6701.

<p>Homology model illustrates thiocarbamylation of catalytic nucleophile Cys126 after treatment with AM6701.</p

Concentration dependent inhibition of purified hNAAA by three compounds.

<p>hNAAA was incubated with the compounds AM6701 (squares), <i>N-</i>Cbz-serine β-lactone (circles), and AM9023 (diamonds) for two hours in order to reach full inhibition before measuring activity. Panel (A). A radioactivity-based assay with [¹⁴C] PEA as substrate. Panel (B). A fluorescence-based assay with PAMCA as substrate. Representative curves are displayed.</p