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dc.contributor.authorKaraveg, Khanita
dc.description.abstractMammalian class 1 .1,2-mannosidases play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally misfolded glycoproteins during ER-associated degradation. Despite several recent reports of X-ray structures of class 1 mannosidases, the proposed catalytic mechanism has not yet been experimentally investigated. As a potential target for therapeutic intervention in ER storage disorders, human ER mannosidase I was chosen to investigate the impacts of single and double mutants of three putative catalytic and two glycone binding residues. The kinetics of binding for a D463N mutant to Man9GlcNAc2 was analyzed by surface plasmon resonance indicating that this residue is mainly responsible for substrate binding, but not catalysis. The optimum pH and pKa shift observed in the E330Q/A mutants strongly indicate that E330 is the general acid catalyst. A proton inventory study gave a DIE of 1.8±0.2, but did not resolve the involvement of a second water residue previously proposed from X-ray structure studies. The presumed general base catalyst is E599 based on X-ray structure determination of a co-complex between an .1,2-mannobiose thiodisaccharide substrate analog and human ER mannosidase I resolved to 1.4 Å. The uncleaved thiodisaccharide co-complex 3bridges the enzyme +1 and -1 subsites and reveals a unique S1 sugar ring conformation for the -1 subsite residue. This information, in combination with prior X-ray structure data of human ER mannosidase I in a co-complex with the glycone mimic, 1-deoxymannojirimycin, suggests that 3the class 1 mannosidases employ a novel H4 sugar conformation in the -1 subsite at the catalytic transition state. Potential roles for additional residues adjacent to the catalytic carboxyl side chains are also proposed to influence the ionization state during acid/base catalysis.
dc.subjectconformational itinerary
dc.titleStructure and function of ER class 1 alpha-mannosidase
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorKelley Moremen
dc.description.committeeKelley Moremen
dc.description.committeeJ. Michael Pierce
dc.description.committeeBi-Cheng Wang
dc.description.committeeWilliam Lanzilotta
dc.description.committeeMarcus Fechheimer

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