The context is, that the inhibitor binds to the viral protease inside the host cell (virus has entered the host cell) and prevents viral replication. In the catalytic site, there is one covalent bond formed and few other hydrogen bonding interaction. Being a covalent bond formation, how will the inhibitor be released? How could one calculate the resident time in such a situation?
Thanks,
Saif
Covalent bonds are not always irreversible. The inhibitor may be able to dissociate even though it has formed a covalent bond. Examples of this are the beta-lactamase inhibitor avibactam and the oxaborole leucyl tRNA synthetase inhibitors. The residence time will be related to the dissociation rate constant. If the rate of replacement of the protein by resynthesis is faster than the dissociation rate, then the residence time will be related to the rate of replacement instead.
If the bond is irreversible, then the inhibitor will remain attached to the protein. The protein will eventually be degraded and replaced by newly synthesized protein. The residence time will be related to the rate of replacement.
Here are some references that show the techniques:
Article Avibactam is a covalent, reversible, non- -lactam -lactamase inhibitor
Article Time-dependent, reversible, oxaborole inhibition of Escheric...
Article Reversibility of Covalent, Broad-Spectrum Serine β-Lactamase...
First, you have to show that the inhibition is covalent. A straightforward way to do that is to show that the inhibitor forms an adduct with the protein, using intact protein mass spectrometry. This could also be done without mass spectrometry using a radioactive derivative of the inhibitor followed by SDS-PAGE and autoradiography.
Next, you have to show that the bond is reversible. This is done by a "jump-dilution" experiment, in which the recovery of enzyme activity is observed following dilution of the enzyme-inhibitor complex.
An additional question is whether the inhibitor dissociates as the original inhibitor or as a modified product. The "acylation exchange" experiment can be used to see whether the inhibitor is intact when it dissociates.
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