Firefly luciferase can use L-luciferin to produce light

Abstract

l-Luciferin is a competitive inhibitor of firefly luciferase with a Ki 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 PPi provided that luciferase is premixed with inorganic pyrophosphatase (PPi-ase). A flash is emitted if PPi is injected into a mixture of luciferase, l-luciferin, ATP and PPi-ase. The system maintains its responsiveness and emits further flashes of about equal duration and intensity upon repeated additions of PPi. It is proposed that PPi induces a racemization of enzyme-bound l-luciferyl adenylate. The potential usefulness of PPi-dependent intracellular ATP monitoring is discussed. The proposed activation of firefly luciferase by PPi may be part of the regulation of in vivo flashing.