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
-Which statement most accurately describes the binding of PBGN to HMBS? (Note: Assume binding is significantly faster than catalysis.)

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
-Which statement most accurately describes the binding of PBGN to HMBS? (Note: Assume binding is significantly faster than catalysis.)

Quizplus · Accepted Answer

11ecba2d_0f05_1a5e_a3bf_ff145d546ace_MD0008_00 Michaelis-Menten kinetics rely on a reaction model in which free enzyme E and free substrate S reversibly combine to form the enzyme-substrate complex ES. The rate of ES formation is proportional to the rate constant k₁, and the rate of dissociation is proportional to k₋₁. After the ES complex forms, the enzyme catalyzes the formation of the product P. The rate of product formation is proportional to k_cat. The Michaelis constant K_m is defined as a combination of the rate constants: 11ecba2d_0f05_416f_a3bf_3b979c3fac24_MD0008_00 Km=k-1+kcatk1 When binding is significantly faster than catalysis k_cat is negligible, so K_m can be approximated as 11ecba2d_0f05_4170_a3bf_bfe6e538ac72_MD0008_00 Km≈k-1k1 This expression is equal to the dissociation constant K_d, which measures binding affinity. Therefore, when binding is faster than catalysis, K_m is an approximate measure of binding affinity. Specifically, binding affinity decreases with increasing K_m. Table 1 shows the Michaelis-Menten equations for each HMBS variant, with PBGN as the substrate. The number in the denominator of each equation is the K_m for that variant, measured in μM. The K_m of the R167W variant is 1600 μM, which is substantially higher than the K_m of any other variant tested. Therefore, PBGN has a lower affinity for the R167W variant than for any other form of the HMBS enzyme tested. (Choice A) The K_m of PBGN for the wild-type enzyme is 70 μM. Both K132N and V215E have lower K_m values, and therefore PBGN has a higher affinity for these variants than for wild-type. (Choice B) The K132N variant has a higher K_m value (63 μM) than the V215E variant (59 μM), so PBGN has a higher affinity for the V215E variant. (Choice D) The K_m values listed range from 59 μM to 1600 μM, so the affinity of PBGN for the enzyme varies significantly between different forms of the enzyme. Educational objective: For enzymatic reactions in which substrate binding is significantly faster than catalysis, the Michaelis constant K_m is an approximate measure of binding affinity. A low K_m corresponds to high affinity and a high K_m corresponds to low affinity. The K_m value is found in the denominator of the Michaelis-Menten equation.

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