Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide

Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.