Investigation of Osmolyte Effects on FolM: Comparison
with Other Dihydrofolate Reductases
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Abstract
A weak
association between osmolytes and dihydrofolate (DHF) decreases
the affinity of the substrate for the <i>Escherichia coli</i> chromosomal and R67 plasmid dihydrofolate reductase (DHFR) enzymes.
To test whether the osmolyte–DHF association also interferes
with binding of DHF to FolM, an <i>E. coli</i> enzyme that
possesses weak DHFR activity, ligand binding was monitored in the
presence of osmolytes. The affinity of FolM for DHF, measured by <i>k</i><sub>cat</sub>/<i>K</i><sub>m(DHF)</sub>, was
decreased by the addition of an osmolyte. Additionally, binding of
the antifolate drug, methotrexate, to FolM was weakened by the addition
of an osmolyte. The changes in ligand binding with water activity
were unique for each osmolyte, indicating preferential interaction
between the osmolyte and folate and its derivatives; however, additional
evidence provided support for further interactions between FolM and
osmolytes. Binding of the reduced nicotinamide adenine dinucleotide
phosphate (NADPH) cofactor to FolM was monitored by isothermal titration
calorimetry as a control for protein–osmolyte association.
In the presence of betaine (proposed to be the osmolyte most excluded
from protein surfaces), the NADPH <i>K</i><sub>d</sub> decreased,
consistent with dehydration effects. However, other osmolytes did
not tighten binding to the cofactor. Rather, dimethyl sulfoxide (DMSO)
had no effect on the NADPH <i>K</i><sub>d</sub>, while ethylene
glycol and polyethylene glycol 400 weakened cofactor binding. Differential
scanning calorimetry of FolM in the presence of osmolytes showed that
both DMSO and ethylene glycol decreased the stability of FolM, while
betaine increased the stability of the protein. These results suggest
that some osmolytes can destabilize FolM by preferentially interacting
with the protein. Further, these weak attractions can impede ligand
binding. These various contributions have to be considered when interpreting
osmotic pressure results