Passage
Synthesis of coenzyme A (CoA) requires vitamin B₅ as a substrate. In bacteria, vitamin B₅ is synthesized by condensation of β-alanine and pantoic acid. β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC) . Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ. Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage. Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity. Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1) . Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity.
Figure 1 Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate
Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4) :492-503.
-Which experiment could best provide data to support the proposed mechanism of autocleavage described in the passage?

Question

Passage
Synthesis of coenzyme A (CoA) requires vitamin B₅ as a substrate. In bacteria, vitamin B₅ is synthesized by condensation of β-alanine and pantoic acid. β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC) . Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ. Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage. Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity. Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1) . Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity.

Passage Synthesis of coenzyme A (CoA) requires vitamin B<sub>5</sub> as a substrate. In bacteria, vitamin B<sub>5</sub> is synthesized by condensation of β-alanine and pantoic acid. β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC) . Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ. Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage. Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity. Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1) . Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity. <strong>Figure 1</strong> Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4) :492-503. -Which experiment could best provide data to support the proposed mechanism of autocleavage described in the passage? A) Measure the rate of ADC formation after site-directed mutagenesis of Thr57 to alanine in PanD. B) Evaluate PanZ's structure with circular dichroism in the presence and absence of AcCoA. C) Measure the molecular weight of PanD in the absence of PanZ and AcCoA. D) Calculate the molecular weight of ADC and isolate it through size-exclusion chromatography.

Figure 1 Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate
Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4) :492-503.
-Which experiment could best provide data to support the proposed mechanism of autocleavage described in the passage?

Quizplus · Accepted Answer

11ecba2d_0e27_da6d_a3bf_2d0f906fb254_MD0008_00 Enzymatic activity generally requires a specific set of amino acid residues in the active site of the enzyme to catalyze the reaction of interest. Replacing any of these critical amino acids with another amino acid typically results in inactivation of the enzyme. Therefore, the role of a specific amino acid in enzyme activity can be determined by deliberately substituting it with a different amino acid. The researchers hypothesized that the PanD autocleavage mechanism involves a conformational change in PanD that allows Thr57 to cleave the N-terminal portion of the protein from the rest of the chain. If this is correct, mutating Thr57 to alanine will most likely eliminate enzymatic activity whereas if Thr57 is not involved in autocleavage, mutating it will most likely have little effect. (Choice B) Circular dichroism can assess secondary structure (α-helices and β-sheets) in proteins. The passage says that PanD, not PanZ, must form a new β-pleated sheet to facilitate autocleavage. Circular dichroism measurements of PanZ will not reveal any conformational changes in PanD. (Choice C) PanZ-AcCoA is required in the autocleavage mechanism. Therefore, the molecular weight of PanD will not change if PanZ and AcCoA are absent. (Choice D) Size-exclusion chromatography separates proteins by size and could be used to separate ADC from PanD, but it would not explain why the molecular weight changed. Educational objective: Enzymatic activity is dependent on the correct sequence and conformation of specific amino acids in the enzyme active site. Mutating critical amino acids generally inhibits enzymatic function. Deliberately mutating amino acids of interest can help reveal their role in enzymatic function.