- Lithium ion holds peptide substrate in cis conformation (60% cis). Ion dissociates in main assay buffer.
- Enzyme catalyses the conversion of the substrate from its cis isomer to its trans isomer.
- Absorbance of the cis and trans form is different, the change in absorbance is measured.
- Substrate will isomerise on its own at a slower rate as it prefers to be in its more stable trans form (see graph below) and this uncatalysed rate is subtracted. The enzymatic rate constant (kenz(s-1)) of the reaction is determined by fitting the data to a 1st order rate equation.
Time course of cis-trans isomerisation in the presence and absence of PPIase