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
-Both isoforms of destabilase have approximately the same k_cat values for isopeptidase activity at optimal pH, but Figure 1 shows that when 5 μL of each purified enzyme was provided with saturating levels of substrate, the isoforms had different levels of activity. What factor could explain this apparent discrepancy?

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
-Both isoforms of destabilase have approximately the same k_cat values for isopeptidase activity at optimal pH, but Figure 1 shows that when 5 μL of each purified enzyme was provided with saturating levels of substrate, the isoforms had different levels of activity. What factor could explain this apparent discrepancy?

Quizplus · Accepted Answer

11ecba2d_0f1f_3241_a3bf_171c07d65590_MD0008_00 An enzyme's turnover number k_cat represents the number of substrate molecules converted to product per second by each enzyme in solution under saturating conditions. The maximum velocity V_max of an enzyme-catalyzed reaction is k_cat multiplied by the enzyme concentration [E]. Therefore, enzymes at a higher concentration in solution will exhibit a higher V_max than enzymes with the same k_cat value present at a lower concentration. The passage states that isoforms 1 and 2 of destabilase have approximately the same k_cat values for both isopeptidase and glycosidase activities at optimal pH, yet Figure 1 shows that 5 μL of isoform 2 has substantially higher activity than the same volume of isoform 1. The best explanation for this observation is that isoform 2 was present at a higher concentration than isoform 1. This could occur if the E. coli bacteria used to produce each isoform expressed isoform 2 at higher levels than isoform 1. This would result in a greater enzyme concentration in the 5 μL sample of isoform 2 (and therefore a higher V_max) than in the 5 μL sample of isoform 1. (Choice A) Under saturating conditions, all enzyme active sites are bound, so any saturated enzyme operates at V_max. Because both isoforms were saturated, both were operating at V_max at every pH tested. In addition, isoform 2 had more activity than isoform 1. (Choice B) Both isoforms eluted at the same imidazole concentration (300 mM), indicating that they have the same affinity for the column. In addition, isoform 1 had less activity than isoform 2, indicating a lower enzyme concentration. (Choice C) Isoforms 1 and 2 have approximately the same optimal pH (~6) for isopeptidase activity. If isoform 1 were denatured at pH 6, it would be inactive, and pH 6 would not be its optimal pH. Educational objective: An enzyme's turnover number k_cat is the number of reactions each enzyme catalyzes per second under saturating conditions. The maximum velocity V_max of an enzyme-catalyzed reaction is equal to k_catmultiplied by the concentration of enzyme [E]. Two enzymes with similar k_cat values may have different V_max values if one of the enzymes is present at a higher concentration than the other.

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