c) How would mutating Asp 02 to a Lysine influence the Ka of chymotrypsin (incre
ID: 204050 • Letter: C
Question
c) How would mutating Asp 02 to a Lysine influence the Ka of chymotrypsin (increase, decrease, of no change)? Explain your logic below with 1-2 sentences. You may assume that the mutation does not affect the overall structure of chymotrypsin or substrate binding. 14 pts) uhích make hPS· alle fo/enne ar Proton. ut we we hay lys-es d) Why is a rate enhancement of nearly 10,000-fold (Kca/kunca) still observed even when each of three residues of the catalytic triad of chymotrypsin are mutated? (2 pts] be caugeExplanation / Answer
Chymotrypsin is synthesized in the pancreas as a precursor chymotrypsinogen. It is a serine protease and serine is a part of catalytic triad that also includes histidine and aspartate. Serine participates in the charge relay system with Histidine and aspartate. In which -NH group of imidazole ring present in the histidine is inturn hydrogen bonded to the carboxylate group of Aspartate.
Answer 1
Mutation from Aspartate to lysine would results in decrease of Turn over number (Kcat) of an enzyme chymotrypsin. Because for the reaction i.e catalyze by the chymotrypsin, due to the presence of negatively charged aspartate, stabilization of the positive charge Histidine residue occurs in the charge relay system.
And lysine residue that replaced the aspartate after mutation, is a basic amino acid like histidine that may create sterric hinderence in the chymotrypsin and affect the folding of the chymotrypsin.
Aspartate is present in the substrate binding site of chymotrysin, mutation in aspartate residue would result in decrease affinity towards peptide and hence Kcat will decrease.
Answer 2
Rate enhancement of about 10,000 fold (Kcat/Km) would occur if mutation replaces one amino acid residue of particular nature (Like acid, basic or non-polar) to different amino acid but of the same nature as of the replaced amino acid.
Example : if aspartate (acidic residue) is found to be replaced by the glutamate (acidic residue), Histidine (basic residue) gets replaced by Lysine (basic residue) and serine (uncharged polar residue) to threonine (uncharged polar residue).
These kinds of mutation may results in increase in Kcat (Turn over number) and decrease the Km ( substrate concentration at which enzyme shows reaction velocity half of its maximum velocity Vmax), which causes increase in the Kcat /Km ratio i.e specificity constant i.e is a measure of an enzyme catalytic efficiency.
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