The mechanism of action of pyrophosphate in firefly luminescence

Abstract

1. 1. Results are presented which demonstrate that the decrease in light intensity after mixing adenosine triphosphate (ATP), Mg ++, luciferin, and luciferase is due to the formation of an inactive complex of luciferase. The low base-line level of luminescence is presumably a measure of a steady-state equilibrium between the inactive complex and active intermediate. The latter is oxidized in the presence of oxygen to give rise to an excited state, which subsequently decomposes to emit a quantum of light. 2. 2. The inactive complex formation depends upon the presence of Mg ++ ion and a second protein. Inorganic pyrophosphatase, which occurs in high concentrations in the firefly lantern, is particularly effective. 3. 3. Purified luciferase, which contains no pyrophosphatase activity, is not rapidly complexed, thus allowing a high steady-state level of luminescence. 4. 4. The addition of pyrophosphate and triphosphate, after initiating the reaction with ATP, stimulates light production, presumably by decomposing the inactive complex. This effect of pyrophosphate depends upon the presence of the inorganic pyrophosphatase. 5. 5. Inhibitors of pyrophosphatase (Mn ++, Ca ++, and F) prevent the rapid decay of the high light intensity obtained with pyrophosphate. The decay of light intensity under these conditions occurs only after the pyrophosphate is hydrolyzed. 6. 6. Pyrophosphate is a potent inhibitor of the luminescent reaction if added prior to ATP. The extent and duration of the inhibition depends, however, upon the concentration of pyrophosphatase in the reaction mixture. The reversal of the inhibition is apparently due to the liberation of luciferase from an inactive complex with pyrophosphate. 7. 7. The results are discussed in relation to a possible mechanism for controlling the rates of enzyme-catalyzed reactions and luminescence in the intact firefly.