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 to explore the structures determined for the 2010 paper "Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases" by Ivan B. Tomasic, Matthew C. Metcalf, Abigail I. Guce, Nathaniel E. Clark and Scott C. Garman. The starting point are the figures found in this paper. Biochemistry students at Westfield State University recreated these figures in jmol, and revised them after getting feedback from the authors. A special thank you goes to Susan Al Mahrwuth, Samuel J. Butler, Susy Civil, Westin G. Cohen, Allison F. DeVivo, Tyler S. Fassett, Courtney M. Fisher, Kimberly Garcia, Stephanie L. Hardy, Maureen W. Kamau, Sienna R. Kardel, Allyson L. Kress, Julia M. Lahaie, Stephen A. Malerba, Brittany E. Ricci, Kimberly Rosario, Yelena Vynar, and Deanna N. Womack for creating the initial figures and captions. If you are interested to learn how these figures were made, take a look at the discussion page (2nd tab above).

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 (GAL). Schindler disease can occur in infancy or in adulthood and is characterized by the lack on the enzyme alpha N-acetylgalactosaminidase (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 overexpress the defective or missing enzyme of interest heterologously 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 GAL protein that is necessary for breaking glycosidic bonds of 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 NAGAL, a protein with a similar active site and function as GAL. Making a new protein closely related to NAGAL with an active site that matches that of GAL allows doctors to administer a protein that serves the function of GAL but has the antigenicity of NAGAL, which means the body will recognize the protein and not elicit an immune response.

Enzymatic activityEnzymatic activity

The enzymes GAL and NAGAL have almost identical active sites, with all residues conserved except for two. Where NAGAL has an alanine and a serine, GAL has a glutamate and leucine. The two enzymes have the same folds and both function to cleave glycosydic bonds, but with different substrate specificities. The differences in substrate specificity occur because NAGAL has the smaller two residues, resulting in a larger binding pocket allows it to bind to N-acetyl galactosamine, which is larger than galactose.

Galactose vs. N-acetyl-galactosamine

The initial rotating molecule in the 3D browser (scroll down if you don't see it) shows the sugar . If you turn off the spinning (click on the +/- control below the spinning molecule) and hover over the atoms, you can learn which colors represent carbon, nitrogen and oxygen (it will display a string like "[A2G]2000:B.C1 #6538", where the letter after the period gives the element symbol, in this case C for carbon).

Structures shown on this pageStructures shown on this page

3H54: the enyme NAGAL in complex with the sugar N-acetyl galactosamine

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

3LX9: the enyme GAL(SA) in complex with the sugar N-acetyl galactosamine

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

Image:Interconversion_model.pdb: Superposition of 3H54 and 3HG5 to make hypothetical model of binding the "wrong" sugar (bonus figure Y).

Overview of the research, and recreated figuresOverview of the research, and recreated figures

The research is about two related enzymes, GAL and NAGAL. They are found in the same location in the human body (in the lysosome, an acidic organelle responsible for breaking down molecules the cell no longer needs), their primary sequence is 50% identical, they catalyze the same reaction (hydrolysis of α-glycosidic bonds), and they have very similar active sites. However, they differ in substrate specificity (one cleaves the bond with galactose, and the other with N-acetyl glalactosamine). In terms of structure, the backbone conformation is quite similar (Fig. 1 panel B and C: compare and . The researchers asked the following question: Is it possible to turn one enzyme into the other (in terms of reaction catalyzed)? Their hypothesis was that a simple swap of the two amino acids in the active site that are different would accomplish an interconversion of specificities. To test this, they made variants called GAL(SA) and NAGAL(EL), in which one active site has the amino acids of the other active site and vice versa (by swapping the two residues that are different). The data obtained by enzyme kinetics supported their hypothesis; the preference for galactose vs N-acetyl galactosamine is swapped (not shown here, but the data is in their paper). Crystal structures (Fig. 2 panel A and B) show how GAL(SA) is able to bind to either or . Comparing the structures of the NAGAL: N-acetyl galactosamine complex and the GAL(SA): N-acetyl galactosamine complex (Fig. 2 panel D) shows that . You can explore the structural data further by going through the figures below and clicking on the buttons. If during your exploration you get lost or some figures behave strangely, press first to reset the 3D browser and then try again. 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 NAGAL) (use the buttons above to compare with NAGAL) Figure Y (bonus figure) The shape of the active site is often complementary to the molecule it binds to (lock-and-key concept). This figure shows the contacts between bound molecules and active site residues. Contacts are shown as the overlap of Van der Waals spheres around atoms. Slight overlap is shown in yellow while larger overlap is shown in red. When two functional groups form a hydrogen bond, atoms come closer than they would if they interact via Van der Waals interactions, so you expect to see red overlap for hydrogen bonds. Here are the contacts for and . These are observed structures, so the contacts seen explain why they bind to their ligands. In contrast, here are the contacts for two hypothetical models, galactose bound to the NAGAL active site and N-acetyl galactosamine bound to the GAL active site. There are less contacts in the and severe clashes in the (clashes involve the acetyl group of the ligand and residues Glu 203 and Leu 206 of the active site). In fact, experiments show that NAGAL does bind galactose (though much more weakly than N-acetyl galactosamine) while GAL does not bind N-acetyl galactosamine. Figure 2: Structure of GAL(SA) GAL(SA) is derived from GAL 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 GAL(SA) is more similar to NAGAL than to GAL (the data is not shown here, but can be found in the research paper). : in complex with N-acetyl galactosamine Use the buttons to hide the model of the sugar in the figure. This is what a crystallographer would interpret to figure out what is bound in the active site. : in complex with galactose : Superposition with alpha-NAGAL bound to GalNAc

spinqualitylabelsall models Colors used for proteins throughout: green (GAL), blue(NAGAL), yellow (GAL(SA)) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Relevant linksRelevant links

Garman lab web site

Structures on proteopedia associated with the Garman lab

Galactosidase