Browsing by Subject "Hydrolysis"
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Item Insights into the Serpin Inhibitory Mechanism from Structures of Mutant Serpins in the Mechaelis Complex(2008-05-13) Sul, Soon-Hee; Goldsmith, Elizabeth J.The serpins belong to a superfamily of protease inhibitors that employ a unique suicide substrate-like inhibitory mechanism. In this mechanism, target protease becomes acylated at the catalytic serine. Deacylation fails to take place, and the serpins undergo dramatic conformational changes in which the acylated protease is translocated 70Å from one pole of the serpin to the other. However, the factors causing suppression of deacylation are not fully understood. The previously solved Michaelis complex of serpin-1B with trypsin/S195A suggests that the P4 (I350) and P1' (S354) residues on the reactive center loop may be important factors in arresting the hydrolysis reaction, since these residues are involved in interactions with trypsin that are unique to serpins (unlike low molecular weight serine protease inhibitors). Inhibition assay data also show that the inhibitory activity of serpin was reduced by the introduction of a mutation either at the P4 (I350A) or at the P1' (S354A) position. The P4 (I350A) mutation nearly completely abolishes inhibitory activity of serpin-1B toward trypsin. In other words, the mutated P4 residue of serpin-1B acts as a substrate, rather than as an inhibitor. A crystallographic approach was used to understand why the serpin-1B (I350A) becomes a substrate; and, hopefully, to gain insight into the serpin inhibitory mechanism. Similarly, the formed Michaelis complex between trypsin and the serpin-1B (S354A) was also utilized to address the role of this residue in this serpin mechanism. The structural analysis shows that significant conformational changes were observed from the serpin-1B (I350A)/trypsin complex, but not from the serpin-1B (S354A)/trypsin complex. Due to these conformational changes, the special extensive interactions observed in the wild type complex were lost in the mutant complexes, the consequence being the destabilization of the mutant Michaelis complexes and, thus, perhaps destabilizing acylated covalent intermediate. Also, reduced interactions (mostly P-side of the serpin) induce a conformational change on trypsin Gln192, residue that may be important for arresting hydrolysis. Therefore, the stable and tight interactions in the Michaelis complex may be important in arresting deacylation in the serpin inhibitory mechanism.Item Intramembrane Proteolysis Mediated by the gamma-Secretase Complex : Nicastrin Functions as a Substrate Receptor(2006-08-11) Shah, Sanjiv; Yu, GangThe proteolytic processing of proteins within the lipid bilayer, and release of their membrane tethered biologically active fragments, fundamentally controls a growing list of cell signaling events. The gamma -secretase, one of a small family of independently evolved proteases, performs this enigmatic hydrolysis of a peptide bond within the membrane. Remarkably atypical, gamma-secretase activity: (1) requires a complex of proteins that include presenilin, nicastrin, Aph1, and Pen-2; (2) catalyzes the intramembrane cleavage of a broad range of substrates, regulating physiology from neurodevelopment to neurodegeneration. The aim of this thesis is to elucidate the mechanism by which the gamma -secretase recognizes its substrates. I provide evidence that nicastrin, in addition to being a critical component of the complex, plays a major function in substrate recognition. The ectodomain of nicastrin binds the new amino terminus that is generated upon the prerequisite 'shedding' of substrates, thereby recruiting substrates into the gamma -secretase complex. The gamma -secretase complex has been traditionally viewed as a hub for signal transduction of substrates such as Notch and APP. The mechanism by which a broad range of substrates may be recognized and subsequently cleaved, as demonstrated in this thesis, supports a mutually inclusive function as a protease that has evolved to simply dispose transmembrane domains thus controlling the repertoire of a class of proteins present in the membrane.