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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.
-Assuming that product formation is the rate-limiting step for all substrates, which of the following statements regarding the formation of the Hip1 enzyme-substrate (ES) complex is correct?

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.
-Assuming that product formation is the rate-limiting step for all substrates, which of the following statements regarding the formation of the Hip1 enzyme-substrate (ES) complex is correct?

Quizplus · Accepted Answer

11ecba2d_0e91_e67d_a3bf_3f93d2910132_MD0008_00 The Michaelis-Menten constant K_m is the substrate concentration at which half of the reaction's maximum velocity is achieved. It depends on both the rate of substrate binding to the enzyme (ES complex formation) and the rate at which the bound substrate is converted to product (product formation). If the rate of product formation is significantly slower than the rate of ES complex formation, as indicated in the question, then K_m essentially measures an enzyme's affinity for a substrate, or the tendency of an enzyme and a substrate to bind and form a complex. When product formation is rate-limiting, K_m can also be thought of as the substrate concentration at which half of the enzymes in solution are bound. A small K_m indicates a strong ES complex because only a small amount of substrate is required to achieve a large amount of complex formation. On the other hand, a large K_m indicates a weak ES complex that does not readily form. According to Table 1, CSL-174 has a smaller K_m for Hip1 than WKLL-ACC does, so CSL-174 forms a stronger ES complex. (Choice A) CSL-175 has a smaller K_m than WKLL-ACC, which means that WKLL-ACC forms a weaker ES complex. In fact, WKLL-ACC has the highest K_m of all, and therefore forms the weakest complex. (Choice C) CSL-176 has the smallest K_m of the peptides tested, and therefore forms the strongest ES complex. (Choice D) CSL-175 has a smaller K_m than CSL-174, so it forms a stronger complex. Educational objective: The Michaelis-Menten constant K_m is an approximate measure of an enzyme's affinity for a substrate or of the strength of the enzyme-substrate complex. A low K_m represents a high affinity and a strong complex whereas a high K_m indicates a low affinity and a weak complex.

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