RHODOBACTER-SPHAEROIDES PHOSPHORIBULOKINASE - BINARY AND TERNARY COMPLEXES WITH NUCLEOTIDE SUBSTRATE-ANALOGS AND EFFECTORS

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP a nd ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoi chiometric binding of these nucleotide triphosphates in PRK's substrat e site is observed not only with wild-type enzyme but also with D42A a nd D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their subst antial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al, (1994) Biochem istry 33, 9343-9350]. Similarly, PRK forms a stable binary complex wit h the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binar y complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleo tide triphosphate that functions as an alternative PRK substrate, form s a binary complex with enzyme, its fluorescence emission is enhanced and lambda(max) shifted from similar to 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucle otide effector is also enhanced and the lambda(max) shifted from simil ar to 460 to 440 nm. PRK forms stable ternary complexes containing NAD H and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH f luorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between th is novel donor/acceptor pair.