Passage
Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent. Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain. The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence. The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis. Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein. Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1) .
Figure 1 Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production. The results are shown in Figure 2.
Figure 2 Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC) . The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1 Effect of εPKC on ALDH2 Variants
Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1) :1-34.
-Based on Table 1, which pair of amino acid substitutions in ALDH2 would most likely result in the highest NADH production in the absence of εPKC?

Question

Passage
Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent. Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain. The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence. The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis. Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein. Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1) .

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD<sup>+</sup> as the oxidizing agent. Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain. The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence. The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis. Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein. Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1) . <strong>Figure 1</strong> Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD<sup>+</sup>, and then monitoring the rate of NADH production. The results are shown in Figure 2. <strong>Figure 2</strong> Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC) . The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.<strong>Table 1</strong> Effect of εPKC on ALDH2 Variants Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1) :1-34. -Based on Table 1, which pair of amino acid substitutions in ALDH2 would most likely result in the highest NADH production in the absence of εPKC? A) T185N and S279Q B) T185D and S279N C) T185E and S279D D) T185Q and S279E

Figure 1 Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production. The results are shown in Figure 2.

Figure 2 Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC) . The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1 Effect of εPKC on ALDH2 Variants

Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1) :1-34.
-Based on Table 1, which pair of amino acid substitutions in ALDH2 would most likely result in the highest NADH production in the absence of εPKC?

Quizplus · Accepted Answer

11ecba2d_0dfb_e839_a3bf_d3535d9388af_MD0008_00 Phosphorylation, a type of protein post-translational modification, adds a phosphate group to serine, threonine, or tyrosine residues. These residues are neutrally charged at physiological pH, but the addition of a phosphate group confers negative charge. This negative charge can alter the activity of a protein, increasing or decreasing its activity. Substituting an amino acid with a negative charge (glutamate or aspartate) into a protein can mimic the effects of phosphorylation. Table 1 shows that in the absence of εPKC, ALDH2 has half the activity as it does in the presence of εPKC. In addition, mutating certain threonine or serine residues (T185 or S279) to alanine (which cannot be phosphorylated) reduces the activity even when εPKC is present. This indicates that εPKC upregulates ALDH2 activity by phosphorylating T185 and S279. If glutamate (E) or aspartate (D) were substituted into positions 185 and 279, the negative charges they carry would likely mimic the effects of negatively charged phosphate. Therefore, a T185E and S279D mutation would most likely increase ALDH2 activity (via the same effects as phosphorylation) when εPKC is absent, resulting in the highest NADH production. (Choices A, B, and D) Asparagine (N) and glutamine (Q) are similar in structure to aspartate and glutamate, respectively, but are not negatively charged. They would not mimic the effect of phosphorylated serine or threonine, and therefore would likely not result in high NADH production when εPKC is absent. Educational objective: Phosphorylation of serine, threonine, and tyrosine residues adds negative charge to a protein, and often alters its activity. The effect of phosphorylation can be mimicked by the negative charges of aspartate or glutamate substituted at positions where phosphorylation normally occurs.

Passage Aldehyde Dehydrogenase 2 (ALDH2) Is Essential for the Oxidation of of Toxic