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.
-The method used to phosphorylate ALDH2 in Experiment 2 must include εPKC and which other component?

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. -The method used to phosphorylate ALDH2 in Experiment 2 must include εPKC and which other component? A) Inorganic phosphate B) ATP C) Phosphoprotein D) Glucose-6-phosphate

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.
-The method used to phosphorylate ALDH2 in Experiment 2 must include εPKC and which other component?

Quizplus · Accepted Answer

11ecba2d_0dfe_593b_a3bf_4341cf106dad_MD0008_00 Kinases are enzymes that catalyze the transfer of phosphate groups from nucleotide triphosphates (ie, ATP, GTP) to other molecules or from phosphorylated molecules to nucleotide diphosphates (ie, ADP, GDP). According to the passage, εPKC is a kinase that phosphorylates proteins such as ALDH2 at certain serine and threonine residues. Attaching phosphate to a molecule is usually a thermodynamically unfavorable process and must be coupled to a thermodynamically favorable process to provide the necessary energy. Hydrolysis of ATP or GTP is thermodynamically favorable. For this reason, kinases use ATP or GTP as a source of phosphate in the reactions they catalyze. Therefore, in addition to εPKC, ATP must also be present for phosphorylation of ALDH2 to occur. (Choice A) Kinases do not transfer inorganic phosphate to molecules. The phosphate must come from a high-energy nucleotide triphosphate such as ATP or GTP. (Choice C) ALDH2 becomes a phosphoprotein once phosphorylation occurs. Prior to phosphorylation, no phosphoproteins are present in this scenario. (Choice D) Glucose-6-phosphate is produced by hexokinase during glycolysis. It is not a requirement for εPKC activity. Educational objective: Kinases are proteins that transfer phosphate groups from ATP or GTP to other molecules or from phosphorylated molecules to ADP or GDP. For a kinase-catalyzed reaction to proceed, ATP or GTP must be present in solution.

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