Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Dihydrofolate reductase (DHFR) is crucial for one-carbon metabolism and is targeted by the antibiotic trimethoprim.
• Trimethoprim resistance has led to the emergence of structurally distinct type II DHFR enzymes.
• Understanding enzyme behavior in crowded cellular environments is vital due to high macromolecule concentrations.

Purpose of the Study:

• To investigate the kinetics and ligand binding of chromosomal E. coli DHFR and drug-resistant R67 DHFR under cell-like conditions.
• To determine the impact of macromolecular cosolutes on DHFR enzyme activity and substrate/cofactor binding.

Main Methods:

• Progress-curve kinetics and ligand binding assays were performed on purified E. coli DHFR and R67 DHFR.
• Enzymes were studied in the presence of various macromolecular cosolutes simulating cellular environments.
• Computer simulations were used to model ligand-protein interactions.

Main Results:

• Macromolecular cosolutes had varied effects on NADPH oxidation and binding, with some increasing and others decreasing affinity.
• Dihydrofolate (DHF) binding and reduction decreased for both DHFR enzymes in the presence of all cosolutes.
• Weak, transient interactions between ligands and cosolutes, rather than steric hindrance, were identified as the primary cause for reduced binding.

Conclusions:

• Cellular crowding significantly impairs dihydrofolate reductase (DHFR) function, reducing both ligand binding and catalytic rates.
• The net charge of protein cosolutes influenced NADP+ binding, while cosolute size affected DHF binding.
• These findings suggest that the in vivo efficiency of DHFR enzymes may be considerably lower than in vitro measurements indicate.