While working on cold active lipase and endoglucanase I found that both enzymes can tolerate EDTA for 1 hr incubation (stability study of the enzyme). But much of the literature said EDTA acts as an inhibitor for enzymes. Can anyone explain why my worked cold active enzymes tolerate EDTA in spite of inhibition?
Thank you in advance for the valuable replies.
I would suggest to check the Calcium concentration. In some cases when we say EDTA is an inhibitor of an enzyme, it does NOT mean that EDTA is direct inhibitor of the enzyme, but it chelates the calcium and by this way produces inhibition on the calcium-dependent enzymes. Therefore, depending on the calcium concentration and amount of added EDTA, you may get no inhibition, partial or complete inhibition. This explanation may interpret your case.
Thank you Dr. H. Babaei.. In both cases of cold active enzymes, enzymes are stable with Calcium ion at 10-50mM concentration and EDTA (100mM) inhibit only 10% enzyme activity while pre-incubated for 1 hr. Is this character have any importance to any field of science?
Hi there,
EDTA is not specific to Ca2+ but it's able to chelate most of the divalent cation like Mg2+, Zn2+, Mn2+... So maybe the answer is about the cation dependency of the activity you are interested in... If preincubation is done in the cold it tends to freeze the system and it might be than chelation by EDTA is not efficient at low temperature...
As say Hossein I think that in this case chelation of ions doesn't affect the enzymes because their mechanism of reaction may not depend on divalent cations, moreover as well as said Dominique cold affect directly EDTA yielding a low efficient chelation process.
EDTA will cause inhibition of divalent ion requiring enzymes and spare those that are metal-independent.
Some enzymes require divalent ions to be more active. This divalent ions may be important in the tridimentional protein structure required for maximum activity in the active center. Negative charged aminoacids (Glu, Asp) can be together by a positive divalent ion bridge ( coo- +Ca+ -ooc). As collegues said this can be different divalent cation (ie Mg+2). If you want to know the divalent cation requirement of your enzyme, you could make a dialysis against mili Q water, and then adding different ammount of divalent cation in order to be close to optimum concentration required. You do the same with different divalent ions, then you will find which are activators and which are inhibitors (ie. heavy metals). Then you could set optimum concentration of divalent ion and then adding different ammount of EDTA, or EGTA, or 1-10 Phenantrolin, in order to see effect of enzyme activity (by chelatin ion effect). HNS
Thank you all for valuable discussion regarding this. Dr. HNS definitely i will try that.
It is known that EDTA is a very powerful iron chelator. Fe(II)EDTA complexes react with hydrogen peroxide to produce reactive hydroxyl radicals. By the way, the use of EDTA in earlier works was a main wrong reason for believing that hydroxyl radicals are formed in cells. It is possible that presence/absence iron ions is a reason of the EDTA effects in your systems.
I agree with all answers. EDTA is a chelator for divalent cations (Ca2+, Mg2+, Zn2+, mn2+, co2+...). It is not an inhibitor of enzyme and act mostly by decreasing the divalent cations concentrations.
What is the enzyme you use ?
As an exemple, look at kinases which have ATP as substrate. In fact, the real substrate is NOT ATP3- but ATPMg-. If you add EDTA, you will have a competition between ATP and EDTA to fix Mg2+, according to the dissociation constants of both complexes (EDTAMg and ATPMg). So, in this case, inhibition by EDTA is only due to a decrease of the ATPMG- concentration, which is the real substrate.
The proposition of Hector is relevant, but be careful, most of the time, if you use a substrate which interact with divalent cations (as ATP3-) there is trace of Mg2+. The dissociation constant is so low that even after dialyse and EDTA addition, you can still have activity due to the presence of low ATPMg- concentration. Myself I used chelator column to try to purify the substrate and try to supress all divalent cations.
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