Passage

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?

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.

Correct Answer:

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