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The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis. Mtb secretes over 70 serine proteases that may be used as viable drug targets. Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response. Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme. Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo. A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1) .
Figure 1 Schematic design of Hip1 fluorogenic substrates. P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide. Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1 Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1
Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11) :807-815.
-Which of the following substrates yields the most optimal catalytic turnover and catalytic efficiency, respectively?

Question

Passage
The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis. Mtb secretes over 70 serine proteases that may be used as viable drug targets. Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response. Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme. Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo. A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1) .

Figure 1 Schematic design of Hip1 fluorogenic substrates. P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide. Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1 Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1

Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11) :807-815.
-Which of the following substrates yields the most optimal catalytic turnover and catalytic efficiency, respectively?

Quizplus · Accepted Answer

11ecba2d_0e93_1efe_a3bf_1535ff9d0e62_MD0008_00 Catalytic turnover refers to the rate at which an enzyme-bound substrate is converted to product. It is given by the rate constant k_cat and is proportional to the maximum reaction rate V_max. On a Michaelis-Menten plot, catalytic turnover is reflected in the height of the plateau that occurs at high substrate concentrations. A high k_cat (or a high plateau) corresponds to high catalytic turnover. Catalytic efficiency is defined by the ratio 11ecba2d_0e93_1eff_a3bf_afaca8f96834_MD0008_00 kcatKm and is a measure of enzyme specificity. It indicates the rate of catalysis at low substrate concentrations and is proportional to the initial slope of the Michaelis-Menten plot. An enzyme that quickly creates product at low substrate concentrations (high initial slope) is said to be catalytically efficient because only a small amount of substrate is needed to generate substantial activity. A high k_cat, a low K_m, or both may contribute to catalytic efficiency. An enzyme-substrate system may have any combination of high and low efficiency and turnover. In this case, CSL-173 has the highest k_cat (turnover) and CSL-176 has the highest 11ecba2d_0e93_4610_a3bf_430206e9b541_MD0008_00 kcatKm ratio (efficiency). (Choice A) Although CSL-173 has the highest catalytic turnover (largest k_cat), WKLL-ACC has the lowest catalytic efficiency ( 11ecba2d_0e93_6d21_a3bf_5941cb97c7c5_MD0008_00 kcatKm ratio). (Choice B) CSL-176 is the most efficient substrate, but WKLL-ACC has the lowest turnover. (Choice C) This option has the optimal catalytic turnover and catalytic efficiency reversed. Educational objective: Catalytic turnover is represented by k_cat and is proportional to the maximum reaction velocity, which is obtained at high substrate concentrations. Catalytic efficiency is represented by 11ecba2d_0e93_6d22_a3bf_b561ed11cca9_MD0008_00 kcatKm and reflects the ability of the enzyme to work at low substrate concentrations.

Passage The Recent Advent of Multidrug-Resistant Mycobacterium Tuberculosis (Mtb) Has Led