I have seen on literature some purified glycoside hydrolases, without cysteines, that showed their activity increased by addicition of DTT or β-mercaptoethanol on enzimatic assay. What are the possible explanations?
Thiol groups are nucleophiles at alkaline pH, and react with aldehydes forming thiohemiacetals. It is therefore possible that is the enzyme has a hemiacetal intermediate, and its hydrolysis is the rate limiting-step, the thiolate may replace an hydroxyl at the enzyme active site and enhance the product release rate.
Yet all of the above is mere speculation, without specific information about the Enzyme and the glycoside, it is very difficult to provide a better answer, and in any case, experimental data would be required to test any proposal.
Best wishes
Rogelio
Allosteric effect is a possibility here as well. The site of action of these compounds might be far from the active site of the enzyme.
What I've read in literature tends to indicate that DTT has a bigger effect than Mercaptoethanol. This makes me think that since DTT is a derivative of a sugar alcohol but with thiol groups, so it may bond with the protein in a covalent manner and induce a change in conformation that makes the enzyme more active.
Also, given that hydrolisis reactions are equilibriums, if DTT reacts with the sugar to produce a glycosyl thiol this could shift the equilibrium torwards hydrolisis, making the enzyme more effective. This is all speculation tho, and I'm sure it can act as a nucleophile, sugar analogue or reducing agent on multiple sites so there are many possible explainations.
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