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BODY 2 OF 4

Active Site
Beta-secretase's active site is on the lumenal side [20] at the N-terminal. It is more open and less hydrophobic than that of the other human aspartic proteases. A flap or hairpin loop partially covers the cleft [14] and there is a positive charge near the active site due to Arg296 [10]. Conserved active site residues Asp32 and Asp228 [22], and the surrounding H-bond (hydrogen bond) network are in the center of the cleft. The signal sequence asp-ser and thr-gly are also present at the active sites [20]. The carboxyls of the active site are not coplanar. The carboxyl extension is highly ordered-residues 369-376 form beta domain with seven H-bonds to strand 293-299, residues 378-383 form a helix-and conformationally different than the extension region observed for other aspartic proteases [23,24,25,26]. There are three disulfide pairs, two of which fasten both sides of the extension region to the C-lobe. It is likely that the last eight residues form a short stem between the globular catalytic domain and the transmembrane region because they are absent form the electron density map. Furthermore, only the last six of the 21 putative pro residues are visible via electron density map [14], suggesting that the others are mobile and therefore consistent with an unstructured pro segment being displaced from the active site by the inhibitor.

Substrate
Beta-secretase's substrate, APP, is also a class I integral transmembrane protein [6]. It has three known sites of in vivo processing [6]. While there are at lease six alternative spliced forms of m-RNA for APP, the m-RNA encoding for the protein containing 695 amino acids is most prominent in the brain [27].

Both enzyme and substrate co-localize in mammalian cells. N- and O-glycosylation and possible tyrosine sulfation takes place after APP protein synthesis [28]. In an intracellular secretory pathway, when APP is endocytosed into early endosomes (acidic compartment-like lysosomes or endosomes-is required for cleavage) [29], APP cleaves at beta secretase positions asp 1 and glu 11 of the Abeta sequence [9,20]. The enzyme is then rapidly recyled and transported to cell [29].

Inhibitors
Bafilomycin A1, which raises the intravesicle pH of lysosomes and endosomes, has been shown to inhibit APP processing by beta-secretase [30]. Because beta-secretase is structurally homologous to HIV aspartic proteases, HIV protease inhibitors are tested for activity against beta-secretase [31]. To obtain optimum therapeutic potential, inhibitor should have low molecular weight (<700 daltons) and high lipophilicity to penetrate the blood-brain barrier [32].
One transition state inhibitor, OM99-2, is eight residues. It has a Ki = 1.6nM for beta-secretase (See Figures 2 and 3 Below).

 

Figure 2

Figure 3

The crystal structure of beta-secretase, created via molecular replacement methods with human pepsin, which has 22% sequence identity of beta-secretase [33,34,35,36] was generated with this inhibitor bound [9]. OM99-2 is located in substrate binding cleft between NH2 - and COOH-terminal lobes [14] The beta-secretase/ inhibitor complex is 798 residues and consists of chains Abeta enzyme, and chains CD-inhibitor. The enzyme interacts with the inhibitor by two H bonds between two active site aspartate residues, by the hydroxyl of the transition state isotere, and by ten H bonds from various regions of