期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 51, 页码 20479-20484出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1311990110
关键词
beta-galactosylceramidase; lysosomal storage disease; glycosyl hydrolase; pharmacological chaperone therapy
资金
- Wellcome Trust [082961/Z/07/Z, 100140]
- Sir Henry Dale fellowship
- Royal Society [098406/Z/12/Z, UF100371]
- Principal Research Fellowship
- Wellcome Trust [082961/Z/07/Z] Funding Source: Wellcome Trust
- Royal Society [UF100371] Funding Source: Royal Society
Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme beta-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of high-resolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzyme-substrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.
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