Structural locations and functional roles of new subsites S5, S6, and S7 in memapsin 2 (β-secretase)

Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP), leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease. The active site of memapsin 2 has been shown, with kinetic data and crystal structures, to bind to eight substrate residues (P-4-P-4'). We describe here that the addition of three substrate residues from P-7 to P-5 strongly influences the hydrolytic activity by memapsin 2 and these subsites prefer hydrophobic residues, especially tryptophan. A crystal structure of memapsin 2 complexed with a statine-based inhibitor spanning P-10-P-4' revealed the binding positions of P-5-P-7 residues. Kinetic studies revealed that the addition of these substrate residues contributes to the decrease in K-m and increase in k(cat) values, suggesting that these residues contribute to both substrate recognition and transition-state binding. The crystal structure of a new inhibitor, OM03-4 (K-i = 0.03 nM), bound to memapsin 2 revealed the interaction of a tryptophan with the S-6 subsite of the protease.