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Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP), leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease. The active site of memapsin 2 has been shown, with kinetic data and crystal structures, to bind to eight substrate residues (P(4)-P(4)'). We describe here that the addition of three substrate residues from P(7) to P(5) strongly influences the hydrolytic activity by memapsin 2 and these subsites prefer hydrophobic residues, especially tryptophan. A crystal structure of memapsin 2 complexed with a statine-based inhibitor spanning P(10)-P(4)' revealed the binding positions of P(5)-P(7) residues. Kinetic studies revealed that the addition of these substrate residues contributes to the decrease in K(m) and increase in k(cat) values, suggesting that these residues contribute to both substrate recognition and transition-state binding. The crystal structure of a new inhibitor, OM03-4 (K(i) = 0.03 nM), bound to memapsin 2 revealed the interaction of a tryptophan with the S(6) subsite of the protease.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.

The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-LeuAla-Val-Glu-Phe, K(i) = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 A resolution. Uniquely defined in the structure are the locations of S(3)' and S(4)' subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-LeuAla-Ala-Glu-Phe, K(i) = 1 nM). Different binding modes for the P(2) and P(4) side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P(2)' alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P(3)' and P(4)'.

Memapsin 1 is closely homologous to memapsin 2 (BACE), or beta-secretase, whose action on beta-amyloid precursor protein (APP) leads to the production of beta-amyloid (A beta) peptide and the progression of Alzheimer's disease. Memapsin 2 is a current target for the development of inhibitor drugs to treat Alzheimer's disease. Although memapsin 1 hydrolyzes the beta-secretase site of APP, it is not significantly present in the brain, and no direct evidence links it to Alzheimer's disease. We report here the residue specificity of eight memapsin 1 subsites. In substrate positions P(4), P(3), P(2), P(1), P(1)', P(2)', P(3)', and P(4)', the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe, and Trp, respectively, while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp, and Phe, respectively. Other less preferred residues can also be accommodated in these subsites of memapsin 1. Despite the broad specificity, these residue preferences are strikingly similar to those of human memapsin 2 [Turner et al. (2001) Biochemistry 40, 10001-10006] and thus pose a serious problem to the design of differentially selective inhibitors capable of inhibiting memapsin 2. This difficulty was confirmed by the finding that several potent memapsin 2 inhibitors effectively inhibited memapsin 1 as well. Several possible approaches to overcome this problem are discussed.

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].

Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophe nyl azo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role.

The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.