Predicting memapsin 2 (β-secretase) hydrolytic activity.
Memapsin 2 (BACE1, β-secretase), a membrane aspartic protease, functions in the cleavage of brain β-amyloid precursor protein (APP) leading to the production of β-amyloid. Because the excess level of β-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 β-amyloid-generating APP variants. Because more β-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.
Li
Xiaoman
X
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Bo
Huang
H
Zhang
Xuejun C
XC
Hartsuck
Jean A
JA
Tang
Jordan
J
D000465:Algorithms; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D015233:Models, Statistical; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D012044:Regression Analysis; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D009419:Nerve Tissue Proteins; C046788:cerebellin; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human