Subsite Specificity of Memapsin 2 (β-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].

Characterization of α2,6-Sialyltransferase Cleavage by Alzheimer's β-Secretase (BACE1)

“…When mutant substrate ST-LA was used, in which Leu at the P1 position was replaced by Ala, cleavage efficiency was reduced to 40% compared with the wild-type substrate (ST-WT), suggesting that the Leu residue in the ST6Gal I peptide is recognized as a preferable amino acid at the P1 position. This result is consistent with those reported previously by others (21,22); Leu at the P1 position in the APP sequence is recognized preferentially by BACE1.…”

“…BACE1 Cleaved ST6Gal I-derived Peptide between Leu 37 and Gln 38 -We confirmed that the secreted ST6Gal I starts at Glu 41 in vivo as well as in cultured cells (19), but in vitro studies by others (21,22) on BACE1 cleavage site preference showed that Lys 40 residue at the P1 position is not a preferable amino acid for BACE1 cleavage. We therefore analyzed BACE1-dependent cleavage of a peptide substrate, DYEAL-TLQAKEFQMPKSQE, which corresponds to Asp 31 ϳGlu 49 of ST6Gal I sequence.…”

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

“…Another peptidic substrate with a much lower K m has been reported recently (25), and we were able to advance mechanistic studies using this substrate and HPLCbased detection method. Under our assay conditions, the latter substrate was cleaved by BACE with k cat of 0.32 Ϯ 0.02 s Ϫ1 and…”

Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

“…When the ␤-secretase inhibitor OM00 -3 was used, Alexa 488-labeled M2 ED (1 M) was preincubated with OM00 -3 (12.4 M) for 30 min at 37°C before adding to cells. The inhibition constant of OM00 -3 for M2 ED at pH 7.0 was determined using the previously described method (15). The internalization of Alexa 488-labeled recombinant M2 ED also was studied by flow cytometry as follows.…”

“…The Swedish mutation at the P 2 -P 1 positions, from Lys-Met to Asn-Leu, enhances the catalytic efficiency by about 60-fold, increases A␤ production, and manifests an early onset form of AD. The specificity of memapsin 2 for all eight substrate residues has been determined previously (15). Memapsin 2 is glycosylated by three N-linked oligosaccharides (16).…”