However, addition of SAM or sinefungin (and analog of the enzymatic product SAH) caused a large increase in the HTRF signal. CJP702 was derived from BIX-01294, which is an uncompetitive inhibitor with respect to SAM. Therefore, the reason for increased signal upon addition of SAM to the binding assay was thought to be likely due to CJP702 also acting as an uncompetitive inhibitor, and detailed kinetic analysis confirmed this mode of inhibition for CJP702. Interestingly, SAH did not promote an increase in HTRF signal, suggesting that SAH binds to a different conformation. With this mind, the HTRF signal reports on conformational changes due to SAM-pocket binding.
The reconfigured peptide binding assay was then used to measure SAM-pocket binding compounds in a kinetic mode (Figure 2). The signal increases are due to conformational changes, which involve several steps, including binding of SAM to free enzyme, resulting in a conformational change in the enzyme allowing CJP702 binding. The rate-determining step is unknown but the t1/2 from kinetic analysis sets the lower limit on the forward rate constants at ~2.5 min. Uncompetitive inhibitors can be common in certain two-substrate–requiring enzyme classes such as dehydrogenases, and this study suggests that HMTs may also have this property.