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the binding cleft to inhibitor backbone. While H bonds are conserved among Eukaryotic [22,14,37]and HIV [14] aspartic proteases, interactions with sides chains of inhibitor are variable. The potency of this inhibitor depends on unmodified hydroxyl (OH) residues in the proper configuration [10].

Active beta-secretase
Active beta-secretase contains 21 residues of the putative pro region (residues 28p-48p) [13] but without transmembrane and intracellular domains [14]. A preprotein in the cell, constitutive glycoslylation/ N-terminal processing and propeptide cleavage in the Golgi apparatus generates a stable, 70 kD glycosylated enzyme [18]. It is suggested that propeptide processing is an autocatalytic mechanism [18]. Point mutations in beta -secretase yield no APP processing to form beta amyloid [38]. beta -secretase 3-D structure modeling suggests that it can hydrolyze a membrane-anchored polypeptide 20-30 residues from the membrane surface. APP's beta -secretase site is indeed located 28 residues from the plasma membrane, and is therefore a prime candidate for beta -secretase proteolysis [6]. All aspartic proteases have 8 subsites for substrate binding (S1 S2 S3 S4 S1` S2` S3` S4`). For beta -secretase, specificity occurs at the S1` subsite, which prefers small or negatively charged side chains like Ala, Asn, Ser, or no side chain-Gly. Furthermore, it seems that at lease two hydrophobic residues are required in the eight subsites [6]. Subsites S2 and S4 are mostly hydrophilic, however, while S1 and S3 are hydrophobic. Corresponding inhibitor side chains P3 and P1 form hydrophobic contacts with the enzyme. P2 and P1 residues adjacent to the beta -secretase cleavage site are Lys and Met. The inhibitor chain changes directions at P2' and leads P3' and P4' toward protein surface. Because the subsite structures are well-defined, they provide a template for drug design selective for beta -secretase.

Recombinant Protein Production and Purification
The C-terminal residues of APP were fused in an E. coli expression vector. Subsequently, full-length APP was generated from 293 cells transfected with either Swedish of wild-type APP. Purification of beta -secretase from human brain was accomplished by way of a four step process that included an affinity-purification step. The recombinant enzyme (beta -secretase-transfected 293T cells) was purified with cation exchange chromatograpy and with inhibitor affinity matrix, after which an N-terminal sequence was determined. It was established that expression of the recombinant enzyme is accompanied by proteolytic processing [10].

Measurement for Activity
Assays for beta-secretase activity were performed on Triton extracts of P2 membranes from various cell types. Assays were performed at pH4.8 in the presence or absence of cysteine proteinase inhibitor E-64 and serine/ cysteine proteinase inhibitor leupeptin. Results categorized the enzyme, beta -secretase, specifically as an aspartyl protease [10].

Biological Significance
Interestingly, while the activity of beta -secretase in humans is at its highest in the brain [10], the expression of the enzyme is highest in the pancreas. Neither function nor substrate for beta -secretase in the pancreas, however, has been uncovered [13]. In the brain, it highest expressed in the hippocampus, cortex and cerebellum [9] More specifically, beta -secretase is present at high levels in neurons as opposed to glia, suggesting that the primary source of extracellular beta amyloid deposited in amyloid plaques are neurons [20].

Proteinaceous brain deposits, including extracellular amyloid plaques, congophilic amyloid angipathy, and intracellular neurofibrillary tangles [39] are the main components of amyloid deposits in beta amyloid Accumulation of longer species (42-43 residues) of beta amyloid in the brain are important in the pathogenesis of AD neurotoxic effect (See Figure 4).

Figure 4

This long beta amyloid is more abundant in sporadic/ non-familial types of AD [2]. Flemish familial AD, with a mutation in APP, increases production of beta amyloid from APP by BACE 2, a BACE (beta -secretase) related transmembrane aspartyl protease [40] that is not highly expressed in the brain [41,42]. This suggests that beta amyloid production is affected by BACE 2 in AD individuals with the Flemish mutation. Swedish mutation of APP at the beta -secretase site elevates beta amyloid production [6]. Hydrolysis of the Swedish peptide by beta -secretase protein domain has a kcat /km value of approximately 60 times that for the wild type APP peptide [6](See Figure 5 Below). Other implications of APP and neurological dysfunctions can be seen in Down syndrome patients who possesss an extra copy of chromosome 21, which is the location of the APP gene at position 21q21.2 [43].