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
-Which conclusion is LEAST supported by the data in Figure 2?

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
-Which conclusion is LEAST supported by the data in Figure 2?

Quizplus · Accepted Answer

11ecba2d_0f23_02d4_a3bf_23cd9eb48d3a_MD0008_00 Enzymes are proteins that catalyze chemical reactions in biological systems. Different amino acids in an enzyme participate in the reaction to different extents. Some amino acids are required for proper protein folding or catalysis; mutating these amino acids will most likely abolish enzyme activity entirely. Other amino acids may help facilitate activity but are not required for enzyme function. Mutating these amino acids results in either no change or a partial decrease in reaction rate but does not completely eliminate activity. The effect of a particular amino acid on enzymatic activity must be determined experimentally. Figure 2 shows the effects of several mutations on both isopeptidase and glycosidase activities in isoform 2 of the destabilase enzyme: When glutamate at position 14 is mutated to alanine (E14A), isopeptidase activity remains high but glycosidase activity is essentially zero. Therefore, Figure 2 strongly supports the conclusion that this glutamate residue is required for glycosidase activity but not isopeptidase activity (Choice A). When lysine at position 38 is mutated to alanine (K38A), isopeptidase activity is essentially zero whereas glycosidase activity is only slightly diminished. Therefore, Figure 2 strongly supports the conclusion that this lysine residue is required for isopeptidase activity but not glycosidase activity (Choice C). When histidine at position 92 is mutated to alanine (H92A), glycosidase activity remains near 100% of wild-type levels and is higher than the glycosidase activity of any other mutation tested. Therefore, mutating this histidine residue has the smallest impact on glycosidase activity (Choice D). Mutating serine at position 31 (S31A) does not completely abolish either isopeptidase or glycosidase activity, but it does diminish both activities. Therefore, the data do not support the conclusion that this mutation has no effect on enzyme activity. Educational objective: Different amino acids within an enzyme affect the enzyme's activity to different extents. Mutations to amino acids that are critical for proper folding or catalysis typically result in complete loss of activity whereas mutations in less critical amino acids result in small changes or no change in activity. The effects of a mutation must be determined experimentally.

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