To deactivate an endoplasmic membrane enzyme, which is actually the last and key enzyme of a complex enzymatic reaction, I want to use the carboxyl group deletion.But I do not want to cause toxicity or inactivation of other cell components. In fact, I want to make this group water soluble. I know I can use carbodiimide, but is it enough to use it alone? What other points should I pay attention to?
When you said 'deletion' I immediately thought of mutagenesis but it seems as though you are looking for a chemical method. I don't think your proposed approach will work. EDC will attack any carboxyl under the right conditions and will cause extensive crosslinking as (almost) all proteins will have carboxyl groups and (almost) all proteins will have lysines.
I'm not sure if it is feasible in your case, but to selectively modify an enzyme carboxyl group chemically you would need a suicide inhibitor.
@Nick Gee thank you for taking time and answer. Yeah, im looking for a chemical method.
So in general, you think that if I use this method, other cellular proteins will be involved?
A workable method would be to treat the cells with a specific inhibitor of the particular enzyme, if such an inhibitor exists.
@Adam B Shapiro Thank you for your response. There are specific inhibitors for this enzyme as a medicine but I am looking for a new, workable one. I'm thinking about how to remove the carboxyl group because based on studies it is an essential group and the enzyme will be inactivated if it is removed.
Dear @Nick Gee what other points should I pay attention to so that healthy cells are not damaged or less damaged? This enzyme is expressed in cancer cells. In fact, my goal is to inhibit this enzyme and I intend to remove its carboxyl group.
You would need a deep understanding of the mechanism and structure of the enzyme to rationally develop a suicide inhibitor, which is the only way of removing the COOH chemically (in reality, I think you would probably react it with something, rather than delete it, but it depends on the mechanism).
A more realistic approach is that alluded to by Adam, a reversible but very high affinity inhibitor, which ideally would be completely selective for the enzyme of interest (though complete selectivity is almost impossible to achieve). In view of the subcellular localisation, the inhibitor would also have to cross membranes to reach the intended target. The intracellular concentration of the inhibitor would have to be sufficiently high to reduce the activity of the enzyme to zero (again almost impossible for reversible inhibitors). The Km of the natural substrate and its cellular concentration are also important parameters, as the natural substrate will be in competition with your inhibitor. It follows that the higher the affinity of the inhibitor the easier it will be to achieve the required outcome.
I assume that there is a particular active site-located carboxyl group you are trying to target. There are, of course, many carboxyl groups in every protein (all asparate and glutamate side chains and the C-terminus).
An active site carboxyl group may have an unusually strong reactivity with a carbodiimide (for example). This means that proteins with such carboxyl groups are more sensitive to inactivation by carbodiimides than other proteins. At a low carbodiimide concentration, such enzymes may be inactivated, whereas other proteins may be less affected. Complete specificity is probably not possible, however, since there are many enzymes with carboxyl groups in their active sites.
Hi Maryam,
A partial inhibition may be achieved by gene silencing approaches. Chemical inhibitors also may be available. The complete elimination of the activity may require a complete knock of the gene activity. The use of carbodiimide may universally affect all proteins containing carboxylic group
Amal Senevirathne Dear Amal Thank you for your response. My main problem is that the exact crystal structure of the enzyme is still unknown. I checked the PDB, PDBe and even PubChem . I just know it has a critical cofactor, and two crucial amino acids in the active site. Inhibitors are mainly designed based on the type of cofactor. Because I read in an article the carboxyl group plays an important role in determining its function, I think about eliminating it. Based on my knowledge and the response of others, I realized that removing this group with chemicals will cause damage to other proteins... so Im a little confused...
Dear Maryam,
If you have at least a close homolog for your enzyme, you can develop a reasonably accurate PDB model using various molecular modeling software. You can do BLAST search for your AA sequence using PDB database and get the maximum score PDB structures. Then you can study the PDB models that give the highest match for the binding domain of your target protein. By taking one or several PDB structures you can deduce the PDB structure for your protein. The quality check of the deduced PDB structure and refinements also can be done. If you want to eliminate the carboxylic group from the enzyme, you may do it in recombinant enzyme and figure out a way to use it in vitro for your purpose.
Amal Senevirathne Dear Amal, Your suggestion seems workable. I'm familiar with molecular docking and R. Do you think such an idea is feasible for a doctoral project?
In addition, existing drugs to inhibit this enzyme are mainly designed based on its cofactor structure. Of course, I would prefer this inhibition to be chemical using a new compound...
Any more suggestion?
This paper might help you.
https://www.jbc.org/article/S0021-9258(20)80349-4/fulltext
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