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 and ATP gamma S-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 lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 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.
Runquist
J A
JA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Narasimhan
C
C
Wolff
C E
CE
Koteiche
H A
HA
Miziorko
H M
HM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000378:metabolism; D004795:Enzyme Stability; D009243:NAD / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
C095967:2-azido-2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D003497:Cyclic N-Oxides; C096873:adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate); D009243:NAD; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase