Two kinetically distinguishable ATP sites in firefly luciferase.

Results are presented which indicate that firefly luciferase has two catalytically active sites. One site, Km of 1.1 X 10(-4) M ATP, is responsible for the initial flash and is apparently product inhibited for further light production. The second site, Km of 2 X 10(-5) M ATP, catalyzes the continuous low production of light. ATP or AMP is a potent inhibitor of the initial flash when LH2-AMP is used to initiate the light reaction but appears to have no affect on the second site low level light emission. Both sites must be occupied by ATP for the formation of one L-AMP. Thus, ATP appears to function both as a catalytically active substrate and a regulator for light emission.