29 ルシフェラーゼ抗体触媒(口頭発表の部)

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Bioluminescence is one of the most attractive natural phenomena, displayed by various different types of organisms from bacteria to fishes. Recently, considerable attention has been directed to bioluminescence reactions due to potential as highly sensitive, nondestructive analytical tools, particularly for monitoring gene expressions and studying protein-protein interactions (luminescence energy transfer). Luciferases catalyze the bioluminescence reactions that involve the oxidations of the luciferins resulting in light emission. Despite their wide applications, little is known about their molecular mechanisms in bioluminescence reactions. We are interested in applying catalytic antibody technology to explore the molecular mechanisms of bioluminescence reactions in proteins. In this work, we have generated catalytic antibodies that catalyze the oxidation reaction of MCLA (1). Hapten 6 was designed as a transition-state analog for the oxidation. The diol moiety in hapten 6 possessed two distinct features. 1) The hydroxylmethyl group at the C-2 position of indanone with a sp^3 carbon mimics a transition-state for the addition of molecular oxygen to 1. 2) The hydroxy group at the C-3 position mimics a transition-state for the formation of the dioxetanone(4) and is also introduced to avoid the decomposition of the hapten via retro-aldol reaction. Of the 70 monoclonal antibodies specific to 6-BSA, 14 accelerated the bioluminescent oxidation of 1. The rate of the oxidation of 2 by antibody 7D10 followed Michaelis-Menten kinetics; apparent values of Micahaelis constant K_m, and rate constant k_<cat> were 52μM and 0.01min^<-1>, respectively. Remarkably, in the presence of 7D10, the luminescent intensity increased 30 fold over the background level of 1. The kinetic characterization and the catalytic mechanism will be discussed.
天然有機化合物討論会の論文
2002-09-01

29 ルシフェラーゼ抗体触媒(口頭発表の部)

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