Passage
The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis. Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors. Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane. The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation. Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1) .
Figure 1 Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site. The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing. This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed. Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively. They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2) .
Figure 2 Percent cleavage of wild-type and mutant Tcd by autoprocessing
Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002.
-Which of the following statements correctly characterizes the bond that is broken during the reaction catalyzed by the IP6-dependent Tcd protease?

Question

Passage
The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis. Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors. Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane. The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation. Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1) .

Figure 1 Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site. The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing. This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed. Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively. They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2) .

Figure 2 Percent cleavage of wild-type and mutant Tcd by autoprocessing
Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002.
-Which of the following statements correctly characterizes the bond that is broken during the reaction catalyzed by the IP6-dependent Tcd protease?

Quizplus · Accepted Answer

11ecba2d_0de1_f76e_a3bf_21fc0b51e22c_MD0008_00 Tcd exhibits protease activity. Proteases are a class of enzymes that catalyze the hydrolysis of peptide bonds in polypeptide chains. Peptide bonds are synthesized from a condensation reaction between the α-carboxyl group of one amino acid and the α-amino group of another. The amide nitrogen has a lone electron pair that it can donate to the peptide bond, allowing peptide bonds to exist in two resonance structures and giving them significant double-bond character. Just as a wheel cannot rotate about two axles, atoms in a molecule cannot rotate about two bonds. Therefore, peptide bonds restrict rotation and limit the conformations that a protein can adopt. (Choice A) Although peptide bond hydrolysis is thermodynamically favorable, it is kinetically slow in the absence of a protease, so peptide bonds do not degrade rapidly in the absence of an enzyme. (Choice C) The double-bond character of peptide bonds restricts each peptide unit to a single plane. Therefore, peptide units are planar rather than tetrahedral. (Choice D) The nitrogen atom in a peptide bond carries a partial positive charge due to resonance. Educational objective: The peptide bond is a resonance-stabilized, planar structure exhibiting partial double-bond character. Rotation about the bond axis is restricted.

Passage The Bacterium Clostridium Difficile Secretes Protein Toxins TcdA and TcdB