Effect of phosphoric acid on ATP and kinases in vitro

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JimmyTee
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Effect of phosphoric acid on ATP and kinases in vitro

Post by JimmyTee » Mon Mar 14, 2011 10:39 am

Hi all,

I am running some kinase assays. It contains my enzyme, a substrate and some ATP all in a physiological buffer. I am told my the manufacturer that i can halt the reaction by adding phosphoric acid to 0.06%. Can anyone explain exactly what is happening to the ATP in here? Is the acid sequestering the kinase ATP binding site, is there a shift in balance away from ATP to ADP because of the phosphoric acid?

Essentially i need to know the effect so i can determine if it will interferre with a downstream phosphatase reaction.

Biochemistry is not my thing, clearly - i've tried googling and various ewbsites explaining acids etc but have not got a suitable answer.

Any help from anyone would be most appreciated.

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JackBean
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Post by JackBean » Mon Mar 14, 2011 8:23 pm

first of all, it is acid, thus lowering pH either denaturing protein or at least shifting the pH from optimal one. That can also hydrolyse the ATP.
Second, it is PHOSPHORIC acid, AKA containing phosphate, which is for sure competitive inhibitor of the kinase.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

JimmyTee
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Post by JimmyTee » Wed Mar 16, 2011 1:14 pm

Hi,

Thanks, i am adding it in to a high capacitance buffer so will check that the pH doesn't change. If it solely works then by competing for ATP binding then that would be ideal.

Is the H3PO (presumably mostly H3PO4 in such a weak solution) able to competitive for binding with ATP, surely they are structurally very different?

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JackBean
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Post by JackBean » Mon Mar 21, 2011 1:22 pm

sure, if you use buffer, the pH should not change significantly.

It is H3PO4 hydrolyzing to (HPO4)2- at pH around 7, which you probably use, rigth?
Sure ATP has much larger moiety than just phosphate, but the kinase binds specifically the phosphate, so it should definitely work as an inhibitor
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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