A research team led by biochemist Scott Garman at the University of Massachusetts Amherst has for the first time determined the mechanism of one of the cell's "recycling" enzymes, human alpha-galactosidase or alpha-GAL, as it breaks down substances in the lysosome, the cell's recycling center.
The work promises to aid treatment of a rare childhood metabolic disorder, Fabry disease. Patients may survive to adulthood but have compromised kidney function or heart disease, for example, due to lipid buildup in blood vessels, tissues and organs.
In people born with a faulty copy of the GLA gene that codes for the human alpha-galactosidase (α-GAL) enzyme, an oily, waxy lipid known as GB3 builds up to toxic levels that leads to Fabry disease symptoms. The cause is remarkably direct—a defect in a single gene means the body fails to produce one specific protein, which causes metabolic disease. Thus, Garman says, "There is a lot of research interest in this one molecule and how it works." His research team, which includes graduate students Abigail Guce, Nathaniel Clark and technician Eric Salgado, report in the current issue of the Journal of Biological Chemistry, available online now.
To learn the enzyme's basic function, the UMass Amherst team, with others in Russia and Sweden, used X-ray crystallography, a technique for creating three-dimensional images of the 6,500 atoms in the large protein. Seeing three dimensions is critical because these protein/enzymes can only carry out their metabolic "missions" by changing shape. They're like origami papers: Inactive and uninteresting while flat, but when folded they become biologically active. To follow the action, scientists must see the folded shapes.
Specifically, as Garman explains, "We used some crystallographic, molecular biological and chemical tricks to trap and examine the enzyme at different stages. Others have done crystallography on the alpha-galactosidase enzyme, but not in this way." They first isolated the enzyme-bound substrate. Next they stopped action to view the enzyme in its covalent intermediate form, and finally stopped action again as the enzyme was about to release its product.
These steps proved to be the key to isolating and understanding the enzyme's normal mechanism. "This is the first time anyone has seen how this enzyme binds the sugar molecule during the reaction when it is cutting one galactose from substrate," Garman says.