Passage
Human gamma-glutamyl transpeptidase (hGGT) is an enzyme involved in cysteine homeostasis, and its overexpression has been linked to asthma, cancer, and other diseases. It catalyzes the addition of water across a bond to cleave extracellular glutathione (GSH) into glutamate and a dipeptide composed of cysteine and glycine. It is believed to function by the mechanism shown in Figure 1.
Figure 1 Proposed mechanism of hGGT-mediated GSH cleavageResearchers interested in investigating hGGT inhibitors as a treatment for pathologies related to excess hGGT activity performed the following experiments, each containing the same concentration of hGGT.Experiment 1hGGT was incubated with various concentrations of GSH in the presence or absence of 250-μM candidate compound acivicin or Compound 1. However, both candidates were found to be unsuitable as acivicin showed significant neurotoxicity and Compound 1 acted as an activator instead of an inhibitor.
Figure 2 Lineweaver-Burk plot of hGGT activity with respect to GSH in the presence or absence of 250-μM candidate inhibitorsExperiment 2Researchers hypothesized that slight variations in the structure of Compound 1 could result in hGGT inhibition and tested several derivatives, shown in Table 1.Table 1 Kinetic Parameters of hGGT in the Presence or Absence of Several Candidate Inhibitors at 250 μM
Experiment 3To better understand the physiological conditions under which hGGT operates, researchers exposed a cultured cell line expressing hGGT on the cell surface to a solution that mimicked the composition of extracellular fluid, including levels of GSH. They found that in this setup, uninhibited hGGT results in a reaction rate that is approximately ½V_max.
Adapted from Wickham S, Regan N, West MB, et al. Inhibition of human ?-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J. 2013;450(3) :547-57.
-Based on Figure 2, Compound 1 activates hGGT in which of the following ways?

Question

Passage
Human gamma-glutamyl transpeptidase (hGGT) is an enzyme involved in cysteine homeostasis, and its overexpression has been linked to asthma, cancer, and other diseases. It catalyzes the addition of water across a bond to cleave extracellular glutathione (GSH) into glutamate and a dipeptide composed of cysteine and glycine. It is believed to function by the mechanism shown in Figure 1.

Figure 1 Proposed mechanism of hGGT-mediated GSH cleavageResearchers interested in investigating hGGT inhibitors as a treatment for pathologies related to excess hGGT activity performed the following experiments, each containing the same concentration of hGGT.Experiment 1hGGT was incubated with various concentrations of GSH in the presence or absence of 250-μM candidate compound acivicin or Compound 1. However, both candidates were found to be unsuitable as acivicin showed significant neurotoxicity and Compound 1 acted as an activator instead of an inhibitor.

Figure 2 Lineweaver-Burk plot of hGGT activity with respect to GSH in the presence or absence of 250-μM candidate inhibitorsExperiment 2Researchers hypothesized that slight variations in the structure of Compound 1 could result in hGGT inhibition and tested several derivatives, shown in Table 1.Table 1 Kinetic Parameters of hGGT in the Presence or Absence of Several Candidate Inhibitors at 250 μM

Experiment 3To better understand the physiological conditions under which hGGT operates, researchers exposed a cultured cell line expressing hGGT on the cell surface to a solution that mimicked the composition of extracellular fluid, including levels of GSH. They found that in this setup, uninhibited hGGT results in a reaction rate that is approximately ½V_max.
Adapted from Wickham S, Regan N, West MB, et al. Inhibition of human ?-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J. 2013;450(3) :547-57.
-Based on Figure 2, Compound 1 activates hGGT in which of the following ways?

Quizplus · Accepted Answer

11ecba2d_0e48_59f6_a3bf_4fca9ce23354_MD0008_00 Activators can enhance enzyme activity by increasing the turnover number k_cat to achieve a higher maximum rate (V_max), decreasing the Michaelis constant K_m to reach the maximum rate more quickly, or both (increased catalytic efficiency, k_cat/K_m). A decrease in k_cat can be offset by a greater decrease in K_m, and an increase in K_m can be offset by a greater increase in k_cat. Lineweaver-Burk plots display the relationship between the inverse of a reaction rate and the inverse of the initial substrate concentration for enzymatic reactions. This relationship is linear, and V_max and K_m can be determined from the y-intercept and the x-intercept, respectively. Catalytic efficiency and k_cat can also be determined, as k_cat = V_max/[enzyme]_tot, and catalytic efficiency is the ratio k_cat/K_m. However, because Lineweaver-Burk plots relate the inverse of these parameters, the trends shown in the plot represent the inverse of the trends of the enzyme-substrate system. For example, a decrease in the y-intercept corresponds to an increase in V_max, and vice versa. Figure 2 shows that in the presence of Compound 1, the y-intercept decreases; therefore, Compound 1 causes an increase in V_max (Choice D). The passage states that all experiments were carried out at the same enzyme concentration, and therefore V_max directly corresponds to the turnover number k_cat. Because V_max increases in the presence of Compound 1, so does the turnover number. (Choice B) Figure 2 shows a decrease in the magnitude of the x-intercept in the presence of Compound 1, which corresponds to an increase in the Michaelis constant K_m. (Choice C) Figure 2 shows an increase in the slope of the line in the presence of Compound 1, indicating an increased K_m/V_max ratio. This is the same as a decreased V_max/K_m ratio, which is directly proportional to the catalytic efficiency, k_cat/K_m. Educational objective: Lineweaver-Burk plots display the inverse of reaction velocity as a function of inverse substrate concentration. As a result, the trends in Michaelis-Menten parameters are inverted on the graph. An increase in y-intercept corresponds to a decrease in V_max and, if enzyme concentration is constant in all experiments, a decrease in the turnover number k_cat.

Passage Human Gamma-Glutamyl Transpeptidase (HGGT) Is an Enzyme Involved in Cysteine