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.
-The first step of the autoprocessing reaction mechanism described in the passage involves modification of a thiol in the active site by an amino acid functioning as a general base. This action is likely to enhance protease activity by:

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.
-The first step of the autoprocessing reaction mechanism described in the passage involves modification of a thiol in the active site by an amino acid functioning as a general base. This action is likely to enhance protease activity by:

Quizplus · Accepted Answer

11ecba2d_0ddb_8eca_a3bf_3f27b0598c41_MD0008_00 In general, amino acids containing thiol (-SH) or hydroxyl (-OH) groups can act as nucleophiles in biological reactions. Nucleophiles are attracted to the positive charge of atomic nuclei, and the greater the negative charge possessed by a molecule, the greater its nucleophilicity. Deprotonation of the cysteine thiol or the hydroxyl group of serine results in negatively charged sulfur or oxygen atoms and enhances their otherwise weak nucleophilicity. The question states that the mechanism begins with the modification of a thiol group. Cysteine is the only amino acid with a thiol (-SH) in its R group. The question further states that the thiol is modified by an amino acid (histidine, in this case) acting as a general base. Bases are proton acceptors; therefore, histidine must be deprotonating, not protonating, cysteine. The deprotonation of cysteine would likely enhance the rate of hydrolysis by converting a cysteine residue in the active site into an anionic-and more nucleophilic-sulfide residue. This sulfide can donate electrons to the carbonyl carbon of the peptide in a process called nucleophilic attack, ultimately leading to hydrolysis. This class of enzyme, known as a cysteine protease, is commonly employed in protein degradation. (Choices B and D) A general base will deprotonate, rather than protonate, a reaction partner. Although protonation would produce a good leaving group, it would not aid in attacking the carbonyl of a peptide bond. (Choice C) Deprotonation of serine would produce a good nucleophile, and serine proteases are common in nature. However, serine does not have a thiol group. Educational objective: Cysteine and serine frequently act as nucleophiles in enzyme-catalyzed reactions. However, they are relatively weak nucleophiles on their own. Therefore, the mechanisms of these reactions often include a deprotonation step to enhance nucleophilicity.

Passage The Bacterium Clostridium Difficile Secretes Protein Toxins TcdA and TcdB