Passage
Porphyrin biosynthesis is a multistep process that is required for production of porphyrin derivatives such as heme, which is a crucial component of hemoglobin, myoglobin, and the cytochrome cofactors of the electron transport chain. In the first step of this pathway, the enzyme porphobilinogen synthase catalyzes the production of porphobilinogen (PBGN) from two δ-aminolevulinate molecules, as shown in Reaction 1.
Reaction 1In the next step, the monomeric enzyme hydroxymethylbilane synthase (HMBS) combines four PBGN molecules to form hydroxymethylbilane (Reaction 2) . Several additional steps are required to convert hydroxymethylbilane to heme.
Reaction 2Defects in HMBS activity can lead to accumulation of δ-aminolevulinate and porphobilinogen in the liver and blood plasma. The accumulation of these molecules can cause acute intermittent porphyria, a disorder characterized by episodes of severe abdominal pain. Researchers expressed, purified, and assessed several variants of HMBS for enzymatic activity (Table 1) and thermal stability (Figure 1) .Table 1 Michaelis-Menten equations generated from several HMBS variants, with V₀ and V_max measured in nmol/hr and [PBGN] and K_m measured in μM. (Note: All reactions were carried out with 1 μM HMBS.)
Figure 1 Thermal stability of several HMBS variants
-Based on the data in Figure 1, a mutation of which amino acid residue has the greatest impact on HMBS stability?

Question

Passage
Porphyrin biosynthesis is a multistep process that is required for production of porphyrin derivatives such as heme, which is a crucial component of hemoglobin, myoglobin, and the cytochrome cofactors of the electron transport chain. In the first step of this pathway, the enzyme porphobilinogen synthase catalyzes the production of porphobilinogen (PBGN) from two δ-aminolevulinate molecules, as shown in Reaction 1.

Reaction 1In the next step, the monomeric enzyme hydroxymethylbilane synthase (HMBS) combines four PBGN molecules to form hydroxymethylbilane (Reaction 2) . Several additional steps are required to convert hydroxymethylbilane to heme.

Reaction 2Defects in HMBS activity can lead to accumulation of δ-aminolevulinate and porphobilinogen in the liver and blood plasma. The accumulation of these molecules can cause acute intermittent porphyria, a disorder characterized by episodes of severe abdominal pain. Researchers expressed, purified, and assessed several variants of HMBS for enzymatic activity (Table 1) and thermal stability (Figure 1) .Table 1 Michaelis-Menten equations generated from several HMBS variants, with V₀ and V_max measured in nmol/hr and [PBGN] and K_m measured in μM. (Note: All reactions were carried out with 1 μM HMBS.)

Figure 1 Thermal stability of several HMBS variants
-Based on the data in Figure 1, a mutation of which amino acid residue has the greatest impact on HMBS stability?

Quizplus · Accepted Answer

11ecba2d_0f07_b272_a3bf_ab9f7c26f090_MD0008_00 The stability of a protein can be measured by its ability to remain folded as ambient temperature increases. An increase in temperature causes the atoms within a protein to move faster and increases the probability that intramolecular interactions such as hydrogen bonds, ionic attractions, and other stabilizing forces will be disrupted, causing the protein to unfold (denature). Proteins that denature at higher temperatures have stronger intramolecular forces and are more stable. The temperature at which 50% of the proteins in solution become denatured is known as the melting temperature (T_m). A higher T_m indicates a more stable protein. Figure 1 shows the T_m of several different HMBS variants. The wild-type form of the enzyme has a T_m slightly above 60 °C. To determine which mutation has the greatest impact on HMBS stability (T_m), the T_m of each HMBS variant must be compared to the wild-type T_m. The T_m of the C73R variant is approximately 55 °C and is further away from the wild-type T_m than any other variant shown. The C73R designation indicates that the cysteine residue (C) at position 73 was mutated to arginine (R). Therefore, mutating the cysteine residue at position 73 has a greater impact on HMBS stability than any of the other mutations listed. (Choices B, C, and D) The K132N, R167W, and V215E variants have T_m values that are closer to the wild-type value than that of the C73R variant. Therefore, mutating lysine at position 132, arginine at position 167, or valine at position 215 has a smaller impact on stability than mutating cysteine at position 73. Educational objective: Protein stability is determined by the strength of the intramolecular forces that maintain a protein in its folded form. The temperature at which 50% of proteins in solution become denatured (melting temperature T_m) is a measure of protein stability. A higher T_m corresponds to a more stable protein.

Passage Porphyrin Biosynthesis Is a Multistep Process That Is Required for for Production