User:Amy Rumora/Sandbox 1 - Revision history

Amy Rumora at 09:03, 4 May 2010

Amy Rumora — Tue, 04 May 2010 09:03:11 GMT

←Older revision		Revision as of 09:03, 4 May 2010
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	[[Image:APPcleavingMechanism.jpg\|left\|thumb\|600px\| '''Catalytic mechanism of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[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= }}		{{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>.	+	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<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<ref>PMID:12458195</ref>.

Amy Rumora at 09:00, 4 May 2010

Amy Rumora — Tue, 04 May 2010 09:00:56 GMT

←Older revision		Revision as of 09:00, 4 May 2010
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	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.		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.	+	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 that are 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.

Amy Rumora at 08:57, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:57:27 GMT

←Older revision		Revision as of 08:57, 4 May 2010
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	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>.		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.	+	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<ref>PMID:12458195</ref>.

	===Low Barrier Hydrogen Bonds===		===Low Barrier Hydrogen Bonds===
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
-	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref>.	+	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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). Proton NMR and SKIE data suggests that formation of a short, strong hydrogen bond occurs in tetrahedral intermediate (FHA)<ref>PMID:12458195</ref>.
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Amy Rumora at 08:55, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:55:56 GMT

←Older revision		Revision as of 08:55, 4 May 2010
Line 61:		Line 61:
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref>.		Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref>.
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	===Evolutionary conservation in the asparic protease family===		===Evolutionary conservation in the asparic protease family===

Amy Rumora at 08:55, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:55:28 GMT

←Older revision		Revision as of 08:55, 4 May 2010
Line 60:		Line 60:
	===Low Barrier Hydrogen Bonds===		===Low Barrier Hydrogen Bonds===
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
-	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.	+	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref>.

	===Evolutionary conservation in the asparic protease family===		===Evolutionary conservation in the asparic protease family===

Amy Rumora at 08:54, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:54:34 GMT

←Older revision		Revision as of 08:54, 4 May 2010
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	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.		Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.

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	===Evolutionary conservation in the asparic protease family===		===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.		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.

Amy Rumora at 08:53, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:53:38 GMT

←Older revision		Revision as of 08:53, 4 May 2010
Line 61:		Line 61:
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]
	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.		Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.
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Amy Rumora at 08:52, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:52:31 GMT

←Older revision		Revision as of 08:52, 4 May 2010
Line 60:		Line 60:
	===Low Barrier Hydrogen Bonds===		===Low Barrier Hydrogen Bonds===
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''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).	+	Previous studies suggest that aspartic acid proteases may contain [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds]<ref>PMID:11601963</ref>. Proton [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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)<ref>PMID:12458195</ref.

Amy Rumora at 08:41, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:41:36 GMT

←Older revision		Revision as of 08:41, 4 May 2010
Line 61:		Line 61:
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''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).		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).
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Amy Rumora at 08:41, 4 May 2010

Amy Rumora — Tue, 04 May 2010 08:41:09 GMT

←Older revision		Revision as of 08:41, 4 May 2010
Line 61:		Line 61:
	[[Image:LBHB.jpg\|left\|thumb\|300px\| '''Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1''' Modified from Toulokhonova et al. (2003) ''J. Biol. Chem.'' 278 (7): 4582–4589.]]		[[Image:LBHB.jpg\|left\|thumb\|300px\| '''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).		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).
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	===Evolutionary conservation in the asparic protease family===		===Evolutionary conservation in the asparic protease family===