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- 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.The enzyme dihydrofolate reductase (DHFR) normally resides in the cytosol, andDHFR can be imported into mitochondria by appending a mitochondrial signalsequence. However, when this modified DHFR is incubated with methotrexate, whichis a substrate analog that binds tightly to the active site, the modified DHFR is nolonger imported.a) Propose an explanation for why methotrexate prevents import of DHFR intomitochondria.b) Suppose that DHFR were modified instead by appending a nuclear localizationsignal. Would you expect methotrexate to prevent transport of this modified DHFRinto the nucleus? Why or why not?Why does it make good physiological sense that regular bouts of prolonged exercise will result in mitochondrial biogenesis?
- For the mitochondrial membrane described in pic, how many protons must be transported to provide enough free energy for the synthesis of 1 mol of ATP (assuming standard biochemical conditions)?The catalytic mechanism of an enzyme found in the mitochondrial matrix (pH = 7.8) depends on an active site Lys residue (pKa = 9.5) that acts as a general acid. Mutation of a neighboring residue alters the microenvironment of the Lys side chain so that its pKa shifts from 9.5 to 7.2. Would the mutation cause the reaction rate to increase or decrease? Explain your answer.The catalytic mechanism of an enzyme found in the mitochondrial matrix (pH = 7.8) depends on an active site Lys residue (pKa = 9.5) that acts as a general acid. Mutation of a neighboring residue alters the microenvironment of the Lys side chain so that its pKa shifts from 9.5 to 7.4. (a) Calculate the ratio of protonated to deprotonated Lys at pH 7.8 for the normal and mutant enzyme. (b) Would you expect the mutation to increase or decrease the reaction rate? Explain your answer.(c) Lys is a classified as a basic amino acid, but it commonly acts as an acid catalyst in biochemical reactions. Explain this paradox.
- In the mitochondrial disease MNGIE a deficiency of thymidine phosphorylase causes dTTP to accumulate in mitochondria. Describe the mechanism by which this occurs. A secondary effect is depletion of mitochondrial dCTP pools. Describe a plausible mechanism for this effect.Leigh syndrome is characterized by psychomotor regression: that is, the progressive loss of mental andmovement abilities. Patients also suffer from lacticacidosis, a condition in which mitochondrial respiration is deficient, so their tissues metabolize glucoseanaerobically, leading to the buildup of lactate. Somepatients with Leigh syndrome have a mutation in themitochondrial gene MT-CO3, which encodes a subunit of the electron transport complex cytochromec oxidase. Other patients diagnosed with Leigh syndrome have a loss-of-function mutation in the nucleargene SURF1, which encodes a factor needed for theassembly of this same enzyme complex.a. How can the same symptoms result from mutationsin a mitochondrial gene and from mutations in anuclear gene?VDAC1 mitochondrial protein plays an important role in human diseases and many strategies can be taken to utilize the therapeutic potential of VDAC1. what are these diseases and strategies?
- Citrate synthase is a nuclear-encoded enzyme of the Citric Acid Cycle. The enzyme is functional in the mitochondrial matrix. Looking at the system required for mitochondrial targeting and import of citrate synthase, which of the following statements are correct? 1. Proper import and processing of citrate synthase is dependent upon other proteins found in the mitochondria. 2. Because of the signal peptide, the mature functional enzyme is actually longer than it would be if it was coded by the organelle genome. 3. Because the citrate synthase signal peptide doesn’t have a precise amino acid signature, mutations that slightly change the primary sequence have no effect on targeting. 4. To get citrate synthase into the mitochondrial matrix requires two signal sequences: one to get it across the outer membrane and a second to get it across the inner membrane. A. 1,2 and 3 B. 1 and 3 C. 2 and 4 D. 4 only E. All of 1,2,3 and 4 are correctExplain the binding of methotrexate interferes with mitochondrial import of the enzyme DHFR which was engineering to carry mitochondrial targeting sequence at its N-terminus.Chambers and coworkers have reported [NAD+] and [NADH] concentrations in yeast mitochondria as 20 mM and 0.3 mM, respectively. Consider the Malate Dehydrogenase reaction below: Malate + NAD+ → Oxaloacetate + NADH + H+ ∆G0’ = +29.7 kJ/mol If Malate concentration in yeast mitochondria is 0.4 mM what is the maximum concentration of oxaloacetate needed to make the reaction exergonic at pH 7.0 and 370C?