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 the information in Experiment 3, if researchers wish to further study the inhibitor that is most potent under physiological conditions, they should choose the one that yields the lowest hGGT activity when GSH concentration is:

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 the information in Experiment 3, if researchers wish to further study the inhibitor that is most potent under physiological conditions, they should choose the one that yields the lowest hGGT activity when GSH concentration is:

Quizplus · Accepted Answer

11ecba2d_0e4a_caf8_a3bf_8f1905c0560e_MD0008_00 The rate of an enzymatically catalyzed reaction is dependent on substrate concentration, which can be expressed in terms of multiples of K_m. The reaction velocity for any enzymatic reaction can be expressed as a percentage of V_max such that if substrate concentration is equal to nK_m, the reaction runs at n/(n+1) V_max. For example, when substrate concentration is equal to K_m, the reaction rate is 1/(1+1) V_max = ½V_max. When substrate concentration is equal to 2K_m, the reaction rate is 2/(2+1) V_max = (2/3)V_max, and so on. The passage states that in a setup that mimics physiological conditions, the reaction runs at approximately ½V_max, and therefore the concentration of GSH under these conditions must be approximately equal to the K_m of the uninhibited enzyme. Some inhibitors are more effective at certain substrate concentrations than others, and an inhibitor that works well under saturating conditions may not work as well when substrate concentration is near K_m. Therefore, if researchers wish to use the inhibitor that is most potent at physiological conditions, they must use the one that works best at the physiological substrate concentration. In this case, they should determine which inhibitor results in the lowest activity when substrate concentration equals the K_m of uninhibited hGGT. (Choice A) A substrate concentration that is orders of magnitude lower than K_m would be significantly lower than physiological concentration, so an inhibitor that works well at this concentration might not work well under physiological conditions. (Choices C and D) A substrate concentration that is four or more times greater than K_m is substantially higher than physiological concentration, so an inhibitor that works well under these conditions might not work well in a physiological setting. Educational objective: Enzymatic reaction rate can be expressed as a fraction of V_max and is directly related to substrate concentration, which can be expressed as a multiple of K_m. Inhibitors that work well at low substrate concentrations ([S] << K_m) may not work as well at higher concentrations, and vice versa.

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