Ja. Runquist et al., RHODOBACTER-SPHAEROIDES PHOSPHORIBULOKINASE - BINARY AND TERNARY COMPLEXES WITH NUCLEOTIDE SUBSTRATE-ANALOGS AND EFFECTORS, Biochemistry, 35(47), 1996, pp. 15049-15056
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.