Scheme 2. A proposed mechanism of the DHS reaction and coupling of the DHS partial reaction with the NADH-Glo reaction.

The DHS complete reaction (DHSRC) consists of two stages DHSR1 and DHSR2. DHSR1 involves two steps: I) NAD-dependent dehydrogenation of spermidine to form an enzyme/dehydrospermidine/NADH complex (EC1), II) cleavage of dehydrospermidine to form an enzyme butylimine intermediate (EI) with bound NADH and the release of 1,3-diaminopropane. DHSR2 involves two steps: III) butylimine transfer from EI to eIF5A precursor to form eIF5A butylimine intermediate/DHS/NADH complex (EC2), IV) reduction of eIF5A butylimine intermediate by enzyme-bound NADH to form deoxyhypusine. The major findings of this study including the release of NADH from EI and its usage in NADH-Glo reaction, and regeneration of free enzyme for cycling of the spermidine cleavage (DHSR1) are indicated by solid black arrows. The DHSR1/NADH-Glo coupled reaction in which enzyme bound NADH is released and used for reduction of proluciferin to luciferin to generate luminescence is shown as a shaded block. The enzyme intermediate has been validated by reduction with NABH₃CN. EC1, putative enzyme complex 1; EI, enzyme intermediate; EC2, putative enzyme complex 2.