The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.
15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.
Alzheimer's disease is characterized by formation of neurofibrillary tangles and amyloid plaques in the regions of the central nervous system that are involved in learning and memory (1). It is believed that accumulation of A 1 in plaques or as soluble aggregates initiates a pathological cascade leading to synaptic dysfunction and neuronal toxicity, with neurodegeneration and dementia as the final outcome (1, 2). Therefore, strategies to reduce the level of brain A are being aggressively pursued as an approach likely to benefit Alzheimer's disease patients. A is produced as the result of sequential proteolysis of a type I transmembrane protein APP by -and ␥-secretases. -Secretase cleaves APP in its extracellular domain at a site close to the membrane surface, a reaction that generates a membrane-bound APP C-terminal fragment of 99 amino acid residues (C99). A subsequent endoproteolysis within the transmembrane domain of C99 by ␥-secretase produces A. Whereas -secretase, an aspartyl protease, has been well characterized (3-6), the identity and structure of ␥-secretase, also thought to be an aspartyl protease (7-12), remains elusive, and its kinetic and catalytic mechanisms are poorly understood. To a large extent, this is due to the highly complicated structural organization of this unusual protease. In contrast to other known proteases, ␥-secretase is composed of a high molecular weight multicomponent complex of transmembrane proteins (13,14). Primarily due to this structural complexity, the catalytic site and mechanism of action of ␥-secretase has not been unequivocally established. Early findings point to presenilin 1 or 2 as the catalytic subunit of ␥-secretase (15, 16). These multipass transmembrane proteins contain two essential aspartate residues in putatively adjacent transmembrane domains (15) and can be cross-linked by high affinity ␥-secretase inhibitors (16 -18). Recent advances have identified three additional proteins, nicastrin (19), aph-1 (20, 21), and pen-2 (20, 22), in the same multicomponent complex, whose co-expression with presenilin appears to be critical for ␥-secretase activity (19 -22). However, the precise roles of these additional protein subunits in the catalytic mechanism of ␥-secretase await further investigation.Associated with the structural complexity of ␥-secretase is the versatility of this protease in cleaving several type I transmembrane proteins. In addition to APP processing, ␥-secretase is required for proteolytic activation of Notch receptor (23-26), a signaling molecule essential for embryonic development of all metazoan species (27). Cleavage of Notch in the transmembrane domain by ␥-secretase generates Notch intracellular domain (NICD), which then translocates into the nucleus, where it regulates gene transcription (28,29). The list of other potential protein substrates for ␥-secretase has recently been expanded to include ErbB4 (30, 31), E-cadherin (32), and CD44 (33). However, the mechanisms by which ␥-secretase reacts with these different substrates remains un...
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