Garman lab: Interconversion of lysosomal enzyme specificities

How this page was createdHow this page was created

The goal of this page is to provide three-dimensional and interactive figures resembling those of an original paper.

Lysosomal storage diseaseLysosomal storage disease

Lysosomal storage disorders are inherited metabolic diseases characterized by an accumulation of undigested various toxic materials. There are nearly 50 diseases and the two examples shown here are Fabry and Schindler disease. Fabry disease, which occurs between early childhood and adolescence, is characterized by the lack of the enzyme alpha galactosidase (α-Gal). Schindler disease can occur in infancy or in adulthood and is characterized by the lack on the enzyme alpha N-acetylgalactosaminidase (α-NaGal). There are currently no cures for lysosomal storage disorders however enzyme replacement therapy is a treatment option. The basic principle of enzyme replacement therapy is to over express the enzyme of interest heterologously, in this case α-Gal α-NaGal, in a cell line and to isolate and purify it from the culture. In enzyme replacement therapy, patients are injected with the enzymes that they lack in the hopes of restoring the enzymatic activity in their cells.

Immune ResponseImmune Response

Individuals suffering from Fabry disease cannot produce the α-GAL protein that is necessary for breaking down Galactose. The usual treatment for this is giving the patient doses of the protein, but this poses a problem. Since the body does not produce the protein, an immune response ranging from severe anaphylaxis to mild discomfort can occur when the patient is given the protein. The body does however produce α-NAGAL, a protein with a similar active site and function as Alpha Gal. Altering the active site of α-NAGAL to match that of α-GAL allows doctors to administer a protein that serves the function of Alpha Gal but has the antigenicity of α-NAGAL, which means the body will recognize the protein and not elicit an immune response.

Enzymatic activityEnzymatic activity

α-Gal and α-NaGal have relatively identical active sites, which are conserved with the exception of alanine, serine, glutamate and leucine which are positioned differently. The two enzymes have the same folds and both function by cleaving glycosydic bonds however have different substrate specificities. The differences in substrate specificity occur because α-NaGal has a larger binding pocket thus interacting with larger molecules but smaller residues.

Galactose vs. N-acetyl-galactosamine

Structures shown on this pageStructures shown on this page

3H54: the enyme α-NAGAL in complex with the sugar GalNAc

3HG5: the enyme α-GAL in complex with the sugar galactose

3LX9: the enyme α-GAL(SA) in complex with the sugar GalNAc

3LXA: the enyme α-GAL(SA) in complex with the sugar galactose

3LXC: the enyme α-GAL(SA) in the presence of glycerol

The initial shows the sugar N-acetyl galactosamine. Figure 1 Panel B (show ) Panel C (show ) Use these buttons to switch back and forth between the two enzymes or to animate the switching If you click on pop-up on the bottom of the 3D browser window, maximize the pop-up window and turn on stereoview (right click on the model, select Style>Stereographic>...), the active site will really pop. Figure X (bonus figure) For the animation in Figure 1, the carbon alpha atoms of the shown active site residues were superimposed (RMSD = 0.3 Å). The following views of the active site differences shows a superposition of the six common carbon atoms (RMSD = 0.02 Å) in the bound sugar. It becomes obvious that the sugar is bound in a slightly different orientation with respect to the overall protein structure. (use the buttons above to compare with α-NAGAL) (use the buttons above to compare with α-NAGAL) Figure 2: Structure of αGAL(SA) αGAL(SA) is derived from αGAL by replacing actives site residues glutamate 203 with serine and leucine 206 with alanine. Having these smaller amino acids in the active site increases the substrate binding cavity, and makes the active site of αGAL(SA) very similar to that of αNAGAL. With these substitutions, the catalytic activity of αGAL(SA) is more similar to αNAGAL than to αGAL (the data is not shown here, but can be found in the research paper). : in complex with GalNAc Crystal structure of α-GAL(SA) bound to GalNAc. α-GAL(SA) active site residues are shown in yellow and the product, GalNAc, is shown in gray. The blue mesh around GalNAc represents its electron density. Use the buttons to hide the model of GalNAc in the figure. This is what a crystallographer would interpret to figure out what is bound in the active site. : in complex with Gal : Superposition with alpha-NAGAL bound to GalNAc Superposition of structures: alpha-GAL(SA) bound to GalNAc, in yellow, and alpha-NAGAL bound to GALNAc, in dark blue. GALNAc product of alpha-GAL(SA) is shown in light brown and GalNAc. product of alpha-NAGAL is shown in light blue. The two product structures are oriented slightly at different angles, but no difference in binding was found. For some reason, this figure breaks figure 1. To get back to figure one, press first.

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