Journal of neurochemistry, 2004-09, Vol.90 (6), p.1312-1320
2004
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- Autor(en) / Beteiligte
- Titel
- Conserved residues within the putative active site of γ‐secretase differentially influence enzyme activity and inhibitor binding
- Ist Teil von
- Journal of neurochemistry, 2004-09, Vol.90 (6), p.1312-1320
- Ort / Verlag
- Oxford, UK: Blackwell Science Ltd
- Erscheinungsjahr
- 2004
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- γ‐Secretase performs the final processing step in the generation of amyloid‐β (Aβ) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for γ‐secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen‐bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue γ‐secretase inhibitor. Here we report that replacement of either tyrosine residue alters γ‐secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Aβ(42) peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0022-3042eISSN: 1471-4159DOI: 10.1111/j.1471-4159.2004.02596.xTitel-ID: cdi_crossref_primary_10_1111_j_1471_4159_2004_02596_x
- Format
- –
- Schlagworte
- Alzheimer's disease, Amyloid beta-Peptides, Amyloid beta-Protein Precursor - metabolism, Amyloid Precursor Protein Secretases, Aspartic Acid - chemistry, Aspartic Acid - genetics, Aspartic Acid - metabolism, Aspartic Acid Endopeptidases - pharmacology, aspartyl protease, Binding Sites - drug effects, Binding Sites - physiology, Biological and medical sciences, Blotting, Western - methods, Cell Line, Cell Membrane - drug effects, Cell Membrane - metabolism, Cell-Free System, Cholic Acids - pharmacology, Chromosomal Proteins, Non-Histone - chemistry, Chromosomal Proteins, Non-Histone - metabolism, Conserved Sequence - physiology, Culture Media, Conditioned - analysis, Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases, Detergents - pharmacology, Embryo, Mammalian, Endopeptidases - chemistry, Endopeptidases - genetics, Endopeptidases - metabolism, Enzyme Activation - drug effects, Enzyme Activation - physiology, Enzyme Inhibitors - pharmacology, Humans, Kidney, Medical sciences, Mutagenesis, Site-Directed - physiology, Neurology, Peptide Fragments, presenilin, Protein Binding - physiology, Protein Folding, Protein Structure, Tertiary, site‐directed mutagenesis, Substrate Specificity, Transfection, Tyrosine - chemistry, Tyrosine - genetics, Tyrosine - metabolism, γ‐secretase