N-Alkylated 6′-Aminoluciferins Are Bioluminescent Substrates for Ultra-Glo and QuantiLum Luciferase: New Potential Scaffolds for Bioluminescent Assays

• Published in: Biochemistry (Easton) Vol. 47; no. 39; pp. 10383 - 10393
• Main Authors: Woodroofe, Carolyn C, Shultz, John W, Wood, Monika G, Osterman, Jean, Cali, James J, Daily, William J, Meisenheimer, Poncho L, Klaubert, Dieter H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30-09-2008
• Subjects: Alkylation; Animals; Catalytic Domain; Kinetics; Light; Luciferases - chemistry; Luciferases - metabolism; Luciferases, Firefly - chemistry; Luciferases, Firefly - genetics; Luciferases, Firefly - metabolism; Luminescence; Models, Molecular; Protein Conformation; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi800505u

A set of 6′-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6′ position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6′-aminoluciferin. Determination of Michaelis−Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 × 104 to 332 × 104 relative light units, but in some cases K m (0.73−10.8 μM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate−enzyme binding.