Rhodobacter sphaeroides Phosphoribulokinase: Binary and Ternary Complexes with Nucleotide Substrate Analogs and Effectors
1996; American Chemical Society; Volume: 35; Issue: 47 Linguagem: Inglês
10.1021/bi9619334
ISSN1943-295X
AutoresJennifer A. Runquist, Chakravarthy Narasimhan, Carl Wolff, Hanane A. Koteiche, Henry M. Miziorko,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoRhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATPγS-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343−9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and λmax shifted from ∼557 to 545 nm. Upon formation of a binary PRK−NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the λmax shifted from ∼460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.
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