Predicting memapsin 2 (β-secretase) hydrolytic activity

Memapsin 2 (BACE1, beta-secretase), a membrane aspartic protease, functions in the cleavage of brain beta-amyloid precursor protein (APP) leading to the production of beta-amyloid. Because the excess level of beta-amyloid in the brain is a leading factor in Alzheimer's disease (AD), memapsin 2 is a major therapeutic target for inhibitor drugs. The substrate-binding cleft of memapsin 2 accommodates 12 subsite residues, from P-8 to P-4'. We have determined the hydrolytic preference as relative k(cat)/K-M (preference constant) in all 12 subsites and used these data to establish a predictive algorithm for substrate hydrolytic efficiency. Using the sequences from 12 reported memapsin 2 protein substrates, the predicted and experimentally determined preference constants have an excellent correlation coefficient of 0.97. The predictive model indicates that the hydrolytic preference of memapsin 2 is determined mainly by the interaction with six subsites (from P-4 to P-2'), a conclusion supported by the crystal structure B-factors calculated for the various residues of transition-state analogs bound to different memapsin 2 subsites. The algorithm also predicted that the replacement of the P-3, P-2, and P-1 subsites of APP from Val, Lys, and Met, respectively, to Ile, Asp, and Phe, respectively, (APP(IDF)) would result in a highest hydrolytic rate for beta-amyloid-generating APP variants. Because more beta-amyloid was produced from cells expressing APP(IDF) than those expressing APP with Swedish mutations, this designed APP variant may be useful in new memapsin 2 substrates or transgenic mice for AD studies.