Rhodobacter sphaeroides phosphoribulokinase: binary and ternary complexes with nucleotide substrate analogs and...

Area of Science:

• Biochemistry
• Enzymology
• Protein-ligand interactions

Background:

• Phosphoribulokinase (PRK) is a key enzyme in carbon fixation.
• Understanding PRK's substrate and allosteric binding is crucial for metabolic pathway analysis.

Purpose of the Study:

• To investigate the binding characteristics of substrate analogs and allosteric effectors to PRK.
• To characterize the structural integrity of PRK mutants and their catalytic activity.
• To explore the formation and properties of PRK-ligand complexes using spectroscopic methods.

Main Methods:

• Enzyme kinetics assays
• Spectroscopic analysis (fluorescence spectroscopy)
• Site-directed mutagenesis

Main Results:

• PRK forms stable binary complexes with ATP, ATP analogs (trinitrophenyl-ATP, ATPγS), and the allosteric activator NADH.
• PRK mutants D42A and D169A exhibit intact substrate binding sites despite diminished catalytic activity.
• The allosteric effector AMP displaces NADH but not ATP from PRK.
• Binding of ligands induces changes in fluorescence emission and spectral maxima for trinitrophenyl-ATP and NADH.
• Stable ternary complexes of PRK-NADH-ATP and PRK-NADH-trinitrophenyl-ATP are formed.
• Energy transfer between NADH and trinitrophenyl-ATP in ternary complexes allows spatial separation estimation.

Conclusions:

• PRK possesses robust substrate and allosteric binding capabilities, even in catalytically impaired mutants.
• Ligand binding induces conformational changes detectable by fluorescence spectroscopy.
• The study provides insights into the structural basis of PRK regulation and function.