Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Ribonucleotide reductase (R1) is crucial for DNA synthesis, catalyzing the rate-limiting step.
• R1 activity is regulated by allosteric effectors binding to distinct sites on the protein.
• Understanding these regulatory mechanisms is key to comprehending DNA replication control.

Purpose of the Study:

• To compare the allosteric regulation and effector binding of wild-type mouse R1 protein with a D57N mutant.
• To investigate the functional consequences of mutating the activity site in mouse R1.
• To elucidate the interaction between the activity and specificity sites in R1.

Main Methods:

• Comparative analysis of wild-type and mutant mouse R1 protein.
• Characterization of effector binding affinities and enzyme kinetics.
• Assessment of nucleotide specificity and catalytic activity.

Main Results:

• Wild-type mouse R1 possesses two distinct effector-binding sites: an activity site (for dATP/ATP) and a specificity site (for dATP, ATP, dTTP, dGTP).
• dATP binds with higher affinity to the specificity site than the activity site in wild-type R1.
• The D57N mutation resulted in loss of nucleotide discrimination and reduced dATP affinity at the specificity site, indicating inter-site communication.

Conclusions:

• The D57N mutation disrupts the distinct affinities of the activity and specificity sites for dATP.
• This mutation highlights the functional interdependence of the two effector-binding sites in mouse R1.
• Mouse R1 shares regulatory similarities with its Escherichia coli counterpart.