Binding of 2,2-diphenylpropylamine at the aldehyde site of bacterial luciferase increases the affinity of the reduced riboflavin 5'-phosphate site.

T F Holzman, T O Baldwin

Index: Biochemistry 20(19) , 5524-8, (1981)

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Abstract

We have found a new class of inhibitors of the bacterial bioluminescence reaction, the N,N-diphenylalkylamines and acids. We have studied the action of one of these compounds 2,2-diphenylpropylamine. The amine was competitive with the long-chain aliphatic aldehyde substrate (Ki congruent to 0.1 mM) but caused an increase in the affinity of the enzyme for reduced riboflavin 5'-phosphate (FMNH2). The inhibitor was attached to Sepharose 6B by a bis(oxirane) spacer, and the interactions of bacterial luciferase with the immobilized ligand were analyzed. The binding of luciferase to the immobilized inhibitor was enhanced by FMNH2 and was decreased by decanal. The results of these studies showed that the 2,2-diphenylpropylamine-luciferase complex has an increased affinity for FMNH2. Likewise, the FMNH2-luciferase complex has an increased affinity for 2,2-diphenylpropylamine. The inhibitor also binds to the enzyme-4a-peroxydihydroflavin complex to block the binding of the aldehyde substrate, while binding of the aldehyde substrate to either the free enzyme or the enzyme-4a-peroxydihydroflavin complex blocks binding of 2,2-diphenylpropylamine.