Chapter 3-1. Mechanism of Protease
Recommended reading: 【Biology】 Chapter 3. Cell and Material Metabolism
Q.
In general, the carbonyl group (C=O) in biochemical reactions is the atom attacked by nucleophiles. Therefore, the C=O double bond of the peptide bond (-CONH-) in a polypeptide chain can be hydrolyzed by water. This reaction is very slow, so it requires the help of enzymes, and proteases such as carboxypeptidase catalyze these reactions. For carboxypeptidase to have full activity, it requires a Zn(Ⅱ) metal ion. Based on this fact, answer the following questions.
⑴ Mechanism of Proteases
Figure 1. Mechanism of subtilisin, one of the proteases
① Step 1. The carboxylate terminus of Asp32 nucleophilically attacks the imidazole ring of His64.
② Step 2. As a result, the conjugate base of the imidazole in His64 nucleophilically attacks the OH group of Ser221.
③ Step 3. The alkoxide of Ser221 formed as a result nucleophilically attacks the carbonyl carbon of the protein.
④ Step 4. The protein is broken down into carboxylic acid and amine by the base-catalyzed amide hydrolysis reaction.
⑤ As implied by the term catalysis, the quantities of Asp32, His64, Ser221 do not change before and after the reaction.
⑵ Types of Proteases
① Pepsin: An endopeptidase that recognizes and cleaves the N-terminus of Phe, Trp, Tyr.
② Trypsin: An endopeptidase that recognizes and cleaves the C-terminus of Lys, Arg
③ Chymotrypsin: An endopeptidase that recognizes and cleaves the C-terminus of amino acids with a phenyl group (Phe, Trp, Tyr)
④ Carboxypeptidase: An exopeptidase that recognizes and cleaves the C-terminus of proteins.
⑤ Subtilisin: A nonspecific protease obtained from Bacillus subtilis.
⑶ Role of the Catalyst
① Answer why the reaction rate is faster when Zn(Ⅱ) metal ion is present at the active site of carboxypeptidase.
Figure 2. Mechanism of thermolysin, a type of carboxypeptidase
○ Carboxypeptidase, trypsin, and chymotrypsin recognize the C-terminus within the protein and cleave so that the carboxyl group is exposed.
○ Basically, they use the acid-catalyzed hydrolysis reaction of amide.
Figure 3. Acid-catalyzed hydrolysis reaction of amide
○ Zn(II) speeds up the reaction because it lowers the activation energy by electrostatically fixing the reaction environment.
② Explain the role of histidine in the catalytic mechanism of chymotrypsin where serine nucleophilically attacks the peptide bond.
○ For the same reason as in ①. Histidine, along with lysine and arginine, is a positively charged amino acid that helps to fix the reaction environment like Zn(II).
Input: 2017.09.17 13:46
Modified: 2020.03.14 09:56