Passage
Destabilase, an enzyme found in the saliva of leeches, exhibits at least two activities: isopeptidase activity and glycosidase activity. Isopeptidase activity is believed to prevent blood clotting by hydrolyzing the isopeptide bond between the ε-amino group of lysine and the γ-carboxyl group of glutamate in fibrin monomers. The enzyme's glycosidase activity digests sugars found in the peptidoglycans of bacterial cell walls and may play a role in innate immunity.Two isoforms of destabilase were tagged with a polyhistidine tail and recombinantly expressed in E. coli cells. Both isoforms were then purified by affinity chromatography and eluted by imidazole, which competes with histidine-tagged proteins for column binding. Each isoform eluted efficiently with 300 mM imidazole. Five microliters of each purified enzyme were then provided with saturating levels of substrate and assessed for isopeptidase and glycosidase activity at various pH levels, which allowed the optimal pH of enzymatic activities for each isoform to be identified (Figure 1) . Further studies showed that at optimal pH, both isoforms had approximately the same k_cat values, but isoform 2 had a lower K_m than isoform 1 for both enzymatic activities.
Figure 1 Isopeptidase activity (A) and glycosidase activity (B) of 5 μL of destabilase isoforms 1 and 2 at various pH valuesIsoform 2 was further characterized by mutating specific residues and measuring enzymatic activities at optimal pH. The results are shown in Figure 2.
Figure 2 Isopeptidase and glycosidase activity of isoform 2 of destabilase with various mutations
- The Lineweaver-Burk plot shown above depicts the glycosidase activity of isoform 1. If both isoforms were tested at equal enzyme concentrations, which of the following would describe the appearance of the plot for isoform 2 relative to isoform 1?

Question

Passage
Destabilase, an enzyme found in the saliva of leeches, exhibits at least two activities: isopeptidase activity and glycosidase activity. Isopeptidase activity is believed to prevent blood clotting by hydrolyzing the isopeptide bond between the ε-amino group of lysine and the γ-carboxyl group of glutamate in fibrin monomers. The enzyme's glycosidase activity digests sugars found in the peptidoglycans of bacterial cell walls and may play a role in innate immunity.Two isoforms of destabilase were tagged with a polyhistidine tail and recombinantly expressed in E. coli cells. Both isoforms were then purified by affinity chromatography and eluted by imidazole, which competes with histidine-tagged proteins for column binding. Each isoform eluted efficiently with 300 mM imidazole. Five microliters of each purified enzyme were then provided with saturating levels of substrate and assessed for isopeptidase and glycosidase activity at various pH levels, which allowed the optimal pH of enzymatic activities for each isoform to be identified (Figure 1) . Further studies showed that at optimal pH, both isoforms had approximately the same k_cat values, but isoform 2 had a lower K_m than isoform 1 for both enzymatic activities.

Figure 1 Isopeptidase activity (A) and glycosidase activity (B) of 5 μL of destabilase isoforms 1 and 2 at various pH valuesIsoform 2 was further characterized by mutating specific residues and measuring enzymatic activities at optimal pH. The results are shown in Figure 2.

Figure 2 Isopeptidase and glycosidase activity of isoform 2 of destabilase with various mutations
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The Lineweaver-Burk plot shown above depicts the glycosidase activity of isoform 1. If both isoforms were tested at equal enzyme concentrations, which of the following would describe the appearance of the plot for isoform 2 relative to isoform 1?

Quizplus · Accepted Answer

11ecba2d_0f20_b8e3_a3bf_9d034a45abc7_MD0008_00 Lineweaver-Burk plots are derived from a rearrangement of the Michaelis-Menten equation. They are linear graphs that display the inverse of an enzymatic reaction rate (1/V₀) as a function of the inverse of substrate concentration (1/[S]). The y-intercept represents the reciprocal of the maximum reaction velocity 1/V_max, the x-intercept represents the negative reciprocal of the enzyme's Michaelis constant −1/K_m, and the slope of the line is the ratio K_mSTRONG_V_max. According to the passage, isoforms 1 and 2 of destabilase have approximately the same k_cat values, but isoform 2 has a lower K_m. A decrease in K_m corresponds to a left-shift in the x-intercept because as K_m decreases, the magnitude of −1/K_m increases, moving it farther away from the origin. Therefore, the x-intercept for isoform 2 will be left-shifted relative to isoform 1. V_max is equal to k_cat × [E], where [E] is the total concentration of enzyme. Because both enzyme isoforms have approximately the same k_cat, they will also have similar V_max values if both isoforms are tested at the same [E]. Relative to the slope of the plot for isoform 1, the numerator (K_m) of isoform 2 is decreased but the denominator (V_max) remains constant, yielding a smaller K_m/V_max ratio. Therefore, the plot for isoform 2 will have a decreased slope compared to isoform 1. (Choice B) If the x-intercept were unaffected, then both isoforms would have the same K_m. The passage states that isoform 2 has a lower K_m than isoform 1. (Choices C and D) A right-shift in the x-intercept would indicate an increase in K_m, but the passage says that K_m is decreased in isoform 2 compared to isoform 1. In addition, an unaffected slope would indicate an unchanged K_m/V_max ratio, and an increased slope would indicate an increased K_m/V_max ratio. Educational objective: Lineweaver-Burk plots display enzyme kinetics in linear form. The y-intercept corresponds to 1/V_max, the x-intercept corresponds to −1/K_m, and the slope corresponds to K_m/V_max.

Passage Destabilase, an Enzyme Found in the Saliva of Leeches, Exhibits