Firefly luciferase can use L-luciferin to produce light

Abstract

L-Luciferin is a competitive inhibitor of firefly luciferase with a K(i) between 3 and 4 μM. Furthermore L-luciferin can serve as an alternative substrate for light production. Catalysis of L-luciferin can be observed in the absence of, or at low concentrations of, D-luciferin. The light production from L-luciferin increases slowly (maximal half-time 8 min) to a stable plateau. At low concentrations of enzyme and L-luciferin, maximal light production is about half of that observed at corresponding D-luciferin concentrations. Increasing the concentration of enzyme or L-luciferin reduces the light production relative to that obtained by D-luciferin catalysis. In contrast to the catalysis of D-luciferin the light production from L-luciferin can be effectively stimulated by the addition of PP(i) provided that luciferase is premixed with inorganic pyrophosphatase (PP(i)-ase). A flash is emitted if PP(i) is injected into a mixture of luciferase, L-luciferin, ATP and PP(i) ase. The system maintains its responsiveness and emits further flashes of about equal duration and intensity upon repeated additions of PP(i). It is proposed that PP(i) induces a racemization of enzyme-bound L-luciferyl adenylate. The potential usefulness of PP(i)-dependent intracellular ATP monitoring is discussed. The proposed activation of firefly luciferase by PP(i) may be part of the regulation of in vivo flashing.