title: Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design.
abstract: Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].
authors.affiliation: Protein Studies Program, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Turner
authors.forename: G
authors.initials: G
authors.lastname: Koelsch
authors.forename: L
authors.initials: L
authors.lastname: Hong
authors.forename: P
authors.initials: P
authors.lastname: Castanheira
authors.forename: J
authors.initials: J
authors.lastname: Ermolieff
authors.forename: A K
authors.initials: AK
authors.lastname: Ghosh
authors.forename: J
authors.initials: J
authors.lastname: Tang
authors.forename: P
authors.initials: P
authors.lastname: Castenheira
authors.forename: A
authors.initials: A
authors.lastname: Ghosh
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D015195:Drug Design; D010450:Endopeptidases; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D019151:Peptide Library; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016564:Amyloid beta-Protein Precursor; D009842:Oligopeptides; D019151:Peptide Library; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human