Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• CF1 is a protein complex derived from lettuce chloroplasts.
• It contains 4 subunits and has a molecular weight of 280,000.
• Formycin nucleotides (FTP) are fluorescent analogs of ATP.

Purpose of the Study:

• To investigate the kinetics of association and dissociation of FTP and FDP with CF1.
• To understand the mechanism of nucleotide binding and its alteration upon ATPase activation.
• To explore implications for energy transduction in photophosphorylation.

Main Methods:

• Utilized formycin fluorescence enhancement to study nucleotide binding kinetics.
• Employed MgCl2 to facilitate nucleotide binding to the latent enzyme.
• Monitored FTP binding as a bimolecular process and its displacement by ATP.

Main Results:

• FTP binding to CF1 exhibits a 3-fold fluorescence enhancement and increased polarization.
• The latent enzyme binds 1 mol of ATP or FTP with dissociation constants (Kd) of 10(-7) and 2x10(-7), respectively.
• Upon activation as an ATPase, the rate of FTP binding increases significantly, suggesting lowered kinetic barriers.

Conclusions:

• The binding and dissociation kinetics of FTP and ATP with CF1 were elucidated.
• Activation of CF1 as an ATPase involves modulation of kinetic barriers for nucleotide exchange.
• These findings provide insights into the mechanism of energy transduction in photophosphorylation.