both side-chain oxygen atoms of aspartate 27 at the active site of dihydrofolate reductase form hydrogen bonds with with the pterdine ring of folates. the importance of this interaction was assessed by studying two mutants at this position, asn 27 and ser 27. the dissociation constant of methotrexate was 0.07 nM for the wild type, 1.9 nM for the ser 27 mutant at 25
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The side-chain oxygen atoms of aspartate 27 form hydrogen bonds with the pteridine ring of folates, which is crucial for the enzyme's function. Show more…
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Both side-chain oxygen atoms of aspartate 27 at the active site of dihydrofolate reductase form hydrogen bonds with the pteridine ring of folates. The importance of this interaction was assessed by studying two mutants at this position, Asn 27 and Ser $27 .$ The dissociation constant of methotrexate was $0.07 \mathrm{nM}$ for the wild type, 1.9 nM for the Asn 27 mutant, and 210 nM for the Ser 27 mutant, at $25^{\circ} \mathrm{C}$. Calculate the standard free energy of the binding of methotrexate by these three proteins. What is the decrease in binding energy resulting from each mutation?
$12-13$ If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import?
Shaiju T.
The enzyme dihydrofolate reductase (DHFR) normally resides in the cytosol, and it can be imported into mitochondria by appending a mitochondrial signal sequence. However, when this modified DHFR is incubated with methotrexate, which is a substrate analog that binds tightly to the active site, the modified DHFR is no longer imported. Propose an explanation for this finding.
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