===BACE1===
BACE1 (β-site of APP cleaving enzyme) also called [http://en.wikipedia.org/wiki/Beta_secretase β-Secretase] and memapsin-2 is a 52 kD class I transmembrane [http://en.wikipedia.org/wiki/Aspartic_acid_protease aspartic acid protease] that cleaves the [http://en.wikipedia.org/wiki/Amyloid_precursor_protein Amyloid Precursor Protein (APP)] in a [http://en.wikipedia.org/wiki/Rate-determining_step rate limiting step] that contributes to the accumulation of β-amyloid plaques in [http://en.wikipedia.org/wiki/Alzheimer's_disease Alzheimer’s disease (AD)]. A subsequent cleavage by γ-secretase generates a 40 or 42 amino acid [http://en.wikipedia.org/wiki/Amyloid_beta β-amyloid peptide]. These peptides can form Aβ plaques that may have deleterious effects on neuronal function and contribute to pathologies of AD. Under normal conditions, BACE1 activity generates a monomeric and soluble Aβ peptide that may play a physiological role in decreasing [http://en.wikipedia.org/wiki/Excitotoxicity excitotoxicity] and neurotransmission at glutamatergic synapses. Additionally, α-secretase and γ-secretase cleave APP to generate p3 and the carboxy terminal fragment AICD in a non-amyloidogenic pathway. In AD, amyloidogenic pathways become preferential over non-amyloidogenic and Aβ plaques appear under increased levels of BACE1 catalytic activity.

[[Image:amyloidogenic.jpg]]
http://www.bioscience.org

===The structure of BACE1 catalytic core===
{{STRUCTURE_1fkn| PDB=1fkn | SCENE= }}
BACE1 is a bilobal protein that consists of luminal N-lobe and C-lobe anchored to the plasma membrane by a transmembrane domain that exposes a small cytosolic domain. The catalytic core of BACE1, shown here, spans amino acid residues 43-385. BACE1 is a member of the mainly beta class of proteins with a β-barrel architecture. This structure is composed of <scene name='User:Amy_Rumora/Sandbox_1/2zht_2ndary/1'>12 helices and 28 sheets</scene> (shown in green and grey, respectively). Unlike other class I aspartic proteases, insertions and an extension in the C-terminus of BACE1 creates an enlarged C-terminal lobe. <scene name='User:Amy_Rumora/Sandbox_1/Acdf/1'>Insertion helix A and insertion loops C, D, and F</scene> are localized on the surface of the C-lobe adjacent from the N-terminus of bound inhibitors. Four acidic amino acid residues on the F insertion loop form a negatively charged area on the surface of the enzyme. <scene name='User:Amy_Rumora/Sandbox_1/Acdfbe/1'>Insertion loops B and E</scene> are located on the surface of the C-lobe near the C-terminus of bound inhibitors. These BACE1 insertions may form interactions with cell surface components and enlarge the C-terminal surface area to make the active site more accessible to substrates<ref>PMID:11021803</ref>. A 35 residue C-terminal <scene name='User:Amy_Rumora/Sandbox_1/Extension/1'>extension</scene> (359-385) forms an ordered β structure spanning residues 369-376 and a helix between residues 378-383. This structure may form a stem with the transmembrane domain. BACE1 contains three structural disulfide bonds. The positioning of these disulfide bonds within BACE1 varies greatly from the location of disulfide bonds in other Class I aspartic proteases<ref>PMID:10749877</ref>. The three structural disulfide bonds maintain the correct fold of BACE1 but are not involved in BACE1 catalysis<ref>PMID:10887202</ref><ref>PMID:11953458</ref>.

===Structural elements involved in catalysis===
{{STRUCTURE_1w50| PDB=1w50 | SCENE= }}
An aspartic acid dyad is located in the active site between the N- and C- terminal lobes. Active site <scene name='User:Amy_Rumora/Sandbox_1/As_asp_greygreen/1'>Asp32 and Asp 228</scene> are in a coplanar configuration that allows them to coordinate and activate a water molecule. The activated water performs a nucleophilic attack on the peptide carbonyl group of the substrate. Hydrogen bonds between the carbonyl group of the aspartates and the substrate stabilize a geminal diol intermediate. A proton is then transferred from aspartate to the leaving group resulting in peptide bond breakage between Met671 and Asp672 of APP. The products of the hydrolysis reaction leave the active site<ref>PMID:18378702</ref>. The active site of BACE1 is covered by a 10 amino acid flexible antiparallel β-hairpin called the <scene name='User:Amy_Rumora/Sandbox_1/Tyr71/1'>flap</scene>. This flap controls the entrance of substrates into the active site and is in closed conformation in the presence of bound inhibitors and open conformation in the inactive or apo structures. Tyrosine 71 is a conserved residue located at the tip of the flap that undergoes large conformational changes. In apo BACE1 structures, Tyr71 forms intra-flap hydrogen bonds allowing the flap to adopt a more open conformation. In the presence of inhibitor, the phenolic ring of Tyr71 forms weak hydrogen bonds with the substrate causing the flap to adopt a closed conformation. A Cα <scene name='User:Amy_Rumora/Sandbox_1/Apo_and_om99/1'>displacement</scene> of 7Å is visible in the inhibitor bound BACE1 complex in comparison to the apo structure<ref>PMID:15451669</ref>. Additionally, the 10s loop undergoes conformational change in the presence of an inhibitor. This loop may confer specificity for the correct substrate.

===BACE1 as a therapeutic target for AD===
{{STRUCTURE_2zhr| PDB=2zhr | SCENE= }}
BACE1 is a major therapeutic target due it's role in generating Aβ plaques that contribute to AD pathogenesis. There are currently over 70 structures of BACE1 complexed with inhibitors in the protein data bank http://www.rcsb.org/. These studies have not only developed inhibitors that efficiently block BACE1 activity, but also provide a greater understanding of the residues and regions that interact with the substrate in the active site of BACE1. The first inhibitor crystallized in complex with BACE1 was OM99-2. <scene name='User:Amy_Rumora/Sandbox_1/Om99-2_inhibitor/1'>OM99-2</scene> is a transition state analogue inhibitor that is eight residues in length (Glu-Val-Asn-Leu*-Ala-Ala-Glu-Phe) designated P4-P4’ (P4-P3-P2-P1*-P1’-P2’-P3’-P4’). OM99-2 inhibits BACE1 cleavage between the P1*-P1’ bond because of the presence of a hydroxyethylene isotere (*) that cannot be cleaved. The S1 and S3 sites that bind P1 and P3 are hydrophobic pockets<ref>PMID:18695942</ref> wherease sites S2 and S4 contain Arg residues that accommodate residues P2 and P4 of the substrate <ref>PMID:16305800</ref><ref>PMID:11021803</ref>. Additional sites S5-S7 (not shown in this structure) are in the proximity of the insertion helix and may be important for recognition of the substrate <ref>PMID:15628850</ref>. The <scene name='User:Amy_Rumora/Sandbox_1/Om99-2_inhibitor_flap/1'>flap</scene> maintains a closed conformation over OM99-2 through hydrogen bonding between the conserved Tyr71 residue and the substrate at positions P1 and P2' <ref>PMID:15096037</ref><ref>PMID:18378702</ref>. Additionally, a conformational displacement is observed for the <scene name='User:Amy_Rumora/Sandbox_1/Om99-2_inhibitor_10s_loop/1'>10s loop</scene> in the hydrophobic S3 site of the BACE1 active site upon binding of an inhibitor.

===pH dependence of BACE1 activity===
[[Image:Electrostatics.jpg|left|thumb|600px| '''Electrostatic maps of BACE1 structures ranging from pH 4.0-7.0''' Structures obtained from Shimizu et al. (2008) ''Mol. Cell Biol.'' 28 (11): 3663–3671.]]
BACE1 is a pH dependent enzyme with optimal enzyme activity at pH 4.5. At this pH, conformational switching of the active site cleft will accommodate the substrate and electron density of catalytic Wat1 is observed in the crystal structure. BACE1 is active at acidic pHs ranging from pH 5.0 to 4.5. At pHs greater than 5, BACE1 structures are in a closed conformation and cannot undergo a conformational transition to bind the substrate<ref>PMID:18378702</ref>. Tyr71 is in a self-inhibiting conformation partially occupying the S1 site in the active site <ref>PMID:16216580</ref>. Below pH 4.5 crystal structures of BACE1 reveal a disordered Wat1 suggesting that this catalytic water is not consistently located in the active site. It is likely that BACE1 employs pH dependence as a dual regulatory mechanism.

Electrostatic maps of crystal structures obtained by Shimizu et al. show that there is a slight increase in positive charge around the flap (blue). Isolated areas around the active site pocket also increase in positive charge. Although there is no crystal structure of APP bound in the BACE1 active site, these areas are near the substrate pockets described to bind negatively charged residues of the OM99 BACE1 inhibitor. Therefore, electrostatic charge probably only plays a small and localized role in the BACE1 catalytic mechanism.

===Catalytic mechanism of BACE1===
[[Image:APPcleavingMechanism.jpg|left|thumb|600px| '''Catalytic mechanism of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
{{STRUCTURE_2zht| PDB=2zht | SCENE= }}
BACE1 catalyzes the hydrolysis of the APP peptide bond between Met671 and Asp672 through a general acid-base mechanism carried out by two highly-conserved aspartic acid residues and a <scene name='User:Amy_Rumora/Sandbox_1/Ph4_5_wat/1'>catalytic water molecule</scene> that stabilizes a [http://en.wikipedia.org/wiki/Geminal_diol geminol-diol intermediate]. BACE1 employs a bi-iso-uni catalytic mechanism predicted to yield two products from one substrate. The enzyme active site contains two catalytic residues, Asp32 and Asp228. Asp32 acts as a general acid while Asp228 acts as a general base with pKas of 3.81 and 9.48, respectively. The free enzyme (E) is monoprotonated. Upon substrate binding (step 1), the loop closes around the substrate forming a tight FHS complex (steps 2). During the third step, the active site water is activated by the basic Asp228. This nucleophilic water is thought to attack the carbonyl carbon of the peptide bond while the acidic Asp32 residue protonates the carbonyl on the substrate resulting in the formation of a *tetrahedral intermediate* FHA (step 3). Peptide bond breakage and protonation of the leaving amine releases the products (steps 4-5, GHPQ and GHQ) and free enzyme without the active site water (GH) (step 6). Finally, the free enzyme incorporates an active site water restoring catalytic function (step 7)<ref>PMID:12458195</ref>.

Unlike other aspartyl proteases, the solvent kinetic isotope effect (SKIE) on catalytic efficiency was small (Kcat/Km) suggesting that proton transfer steps up to the irreversible step (step 4) are not rate limiting. It is important to note that low catalytic efficiency could also be due to multiple proton transfers offsetting one another, loop closure causing water displacement, or formation of an amide hydrate intermediate. Turnover number (kcat), on the other hand, is greatly effected by solvent showing an inverse SKIE. Formation of the tetrahedral intermediate is not rate limited as indicated by no SKIE on catalytic efficiency. The final step is predicted to be the rate limiting step a recharging step where the enzyme is restored to its initial state by regaining a catalytic water molecule. This is supported by the inverse SKIE on turnover of BACE1.

===Low Barrier Hydrogen Bonds===
[[Image:LBHB.jpg|left|thumb|600px| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
Proton NMR studies of free BACE1 protein shows a downfield signal resonating at δ=11.8 ppm. In comparison, the enzyme complexed to the OM99-2 inhibitor reveals another resonance at δ=13.0 ppm. The D/H fractionation factor for this downfield proton chemical resonance was lower (∅=0.6) than that of free BACE1 (∅=2.2). This and SKIE data suggests that formation of a short, strong hydrogen bond occurs in tetrahedral intermediate (FHA).

===Evolutionary conservation in the asparic protease family===
Members of the aspartic protease family have structural features that are evolutionarily conserved and appear in most aspartic proteases. [[Pepsin]], [http://www.rcsb.org/pdb/explore.do?structureId=1LYB Cathepsin D], [http://www.rcsb.org/pdb/explore.do?structureId=1CZI Chymosin], and [http://www.rcsb.org/pdb/explore.do?structureId=2V13 renin] are members of the aspartic protease family and are evolutionarily related to BACE1. Like BACE1, these proteins have a bilobal structure, a catalytic dyad composed of two Asp, and have a β-barrel architecture. On the other hand, BACE1 has several unique features that separate it from other aspartic proteases. Currently, BACE1 is the only member of this family that is a transmembrane protein. The addition of extension loops and helices enlarge the BACE1 C-lobe and the location of disulfide bonds varies in comparison to other aspartic proteases.

===Links===
*[[1w50]] (Apo structure of bace (beta secretase))
*[[1w51]] (Bace (beta secretase) in complex with a nanomolar non-peptidic inhibitor)
*[[2zhs]], [[2zht]], [[2zhu]], [[2zhv]] (Crystal structure of BACE1 at varying pH)
*[[2zhr]] (Crystal structure of BACE1 in complex with OM99-2 at pH 5.0)
*[[1fkn]] (Structure of Beta-Secretase Complexed with Inhibitor)

===References===
<references />

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