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.
-In further experiments researchers found that bacteria lacking Hip1 (knockout bacteria) were able to cleave WKLL-ACC but not the CSL substrates. How might these results be explained?

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.
-In further experiments researchers found that bacteria lacking Hip1 (knockout bacteria) were able to cleave WKLL-ACC but not the CSL substrates. How might these results be explained?

Quizplus · Accepted Answer

11ecba2d_0e97_64ca_a3bf_e5c24e85ec10_MD0008_00 The passage mentions that over 70 serine proteases are secreted by Mtb. WKLL-ACC yields fairly low Hip1 activity but may have specificity for one of the other proteases. Genetic removal of Hip1 would be a simple way to test which substrates require Hip1 and which work with other proteases. Because the CSL substrates are not strongly cleaved in the absence of Hip1, they are most likely specific to Hip1. In contrast, Hip1 removal had a minimal effect on WKLL-ACC cleavage, suggesting that WKLL-ACC strongly interacts with one or more other proteases but does not interact strongly with Hip1. (Choice A) Although peptide hydrolysis is a thermodynamically favorable process, it is kinetically slow. It does not occur quickly unless an enzyme is present to increase the rate. (Choice B) The serine proteases discussed in the passage are secreted and function outside the cells, so peptide uptake into bacteria is irrelevant to the rate of hydrolysis. (Choice D) Knocking one gene down can indeed result in upregulation of other genes to compensate. However, if the upregulated proteases were nonspecific, they would likely act on the CSL substrates just as readily as they would on WKLL-ACC. Educational objective: Substrates with high specificity for one enzyme may have low specificity for another. Conversely, a substrate with low specificity for an enzyme of interest may be significantly acted upon by another enzyme in a living system. This scenario can be tested by knocking out a gene of interest in a living organism.

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