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
-Given that blood pH is 7.4, what is the approximate isopeptidase activity level of isoform 1 in blood expressed as a percentage of optimal activity?

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
-Given that blood pH is 7.4, what is the approximate isopeptidase activity level of isoform 1 in blood expressed as a percentage of optimal activity?

Quizplus · Accepted Answer

11ecba2d_0f1d_f9c0_a3bf_9f74f5363e31_MD0008_00 Enzymes consist of several amino acids linked together by peptide bonds. Some amino acids contain side chains that can be protonated or deprotonated depending on the ambient pH. As the protonation state of a side chain changes, its ability to stabilize the enzyme's structure or to interact with substrates changes as well. For this reason, an enzyme's activity typically varies with pH, and each enzyme has an optimal pH, which must be determined experimentally. Figure 1 shows that for isopeptidase activity, both isoforms of destabilase have an optimal pH of approximately 6. Isoform 1 converts substrate to product at a rate of ~800 ng/min at optimal pH. As pH increases above 6, isopeptidase activity decreases. The graph shows that at the pH of blood (7.4), the isopeptidase activity diminishes to ~400 ng/min. Therefore, at pH 7.4, isoform 1 exhibits isopeptidase activity that is 50% of its optimal activity (400/800). (Choice A) At optimal pH, Figure 1A shows that isoform 1 has approximately 40% (800/2,000) of the activity of isoform 2 at the same pH. However, the question asks how isoform 1 compares to itself as the pH changes from optimal pH (6) to physiological blood pH (7.4). (Choice C) Figure 1A shows that isoform 2 has approximately 80% of its optimal activity at pH 7.4 (1,600/2,000), but the question asks about isoform 1. (Choice D) Isoform 1 of destabilase exhibits 100% of its optimal glycosidase activity at a pH of 7.4 (Figure 1B), but the question asks for isopeptidase activity. Educational objective: Enzyme activity is dependent on the ambient pH. Changes in pH can alter the protonation states of some amino acid side chains, which can alter their ability to stabilize enzyme structure and interact with substrates. The optimal pH of an enzyme must be determined experimentally.

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