摘要BACKGROUND:β-secretase (β-site APP cleavage rate-limiting enzyme, BACE) has been proposed as a promising therapeutic target for Alzheimer's disease (AD). BACE inhibition reduces production of β-amyloid peptide (Aβ) and promotes neural regeneration. Two catalytic aspartates (Asp 32 and Asp 228) exist in a monoprotonated state in the active BACE site, but the precise proton location remains unclear.OBJECTIVE:To explore the entire process of BACE enzymatic hydrolysis using quantum chemistry calculations, and to identify the precise proton location for Asp 32 and Asp 228 during the enzymatic process.DESIGN, TIME AND SETTING:According to protonation state of BACE, four tautomers were designed and quantum chemistry calculations were performed at the Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, China between October 2008 and March 2009.MATERIALS:Hardware:linux workstation (Department of Equipment, Sun Yat-sen University, China); software:QSITE, Glide, Maestro (Schrodinger LLC, USA), MOPAC 2007 (CAChe Research LLC, USA), Triton 4.0 (National Centre for Biomolecular Research, Czech Republic) were used.METHODS:Using crystal structures of BACE to build a catalytic model (enzyme, catalytic water, and substrate peptide EVNLAAEF) on the computer and superimposition, four BACE tautomers (32i, 320, 228i, and 2280) in the monoprotonated state were developed with Schrodinger package. Hybrid quantum mechanical/molecular mechanic (QM/MM) calculations were performed at the B3LYP density functional theory level to identify the precise proton location for the dyad aspartic residues (Asp 32 and Asp 228). Using the most possible tautomer as the reactant, the entire enzymatic hydrolysis of substrate EVNL/AAEF was simulated at the semiempirical level.MAIN OUTCOME MEASURES:The precise proton location of was measured by analyzing co-planarities of 4 BACE tautorners (32i, 32o, 228i, and 2280) in the monoprotonated state, because the dihedral formed by the carboxyl oxygen atoms of the dyad aspartic residues. The transition state and the production state, as well as activation energies and reaction enthalpies, were measured by calculating geometric and energy changes during catalytic reaction of the system.RESULTS:In the 2280 BACE tautomer, the dihedral angle of the four oxygen atoms in the catalytic aspartates was 8.7°, which was the lowest of four tautomers. The lowest activation energy and highest reaction enthalpy (Ea = 216.30 kJ/mol, AH = 30.98 kJ/mol) were also found in 2280, among the four tautomers during the catalytic reaction. In addition, when the reaction proceeded to the transition state, followed by product generation, the proton location was reversed to the inner oxygen of Asp 32 (32i) from the outer oxygen of Asp 228 (228o).CONCLUSION:Results demonstrated the mechanism of Aβ generation. At beginning of BACE catalytic reaction, the precise proton location was preferred on the outer oxygen of Asp 228 (228o). In this protonation state, catalytic reaction can proceed smoothly, with reduced active energy and heat release. When the reaction proceeded to the transition state and product generation, the proton location was reversed to the inner oxygen of Asp 32 (32i). These results provide theoretical guidance for designing new drugs to protect neural cells and promote neural regeneration in Alzheimer's patients.