Question

Question 11 pts

When chymotrypsin is assayed with the surrogate substrate p-nitrophenylacetate, a rapid burst of colored product formation (p-nitrophenolate) is observed, corresponding to a relatively steep slope on the A410 vs. time (seconds) plot, followed by a slower-but-steady release of p-nitrophenolate, corresponding to a relatively less-steep slope on the A410vs. time (seconds) plot. These results were interpreted as:

a)	the rapid release of the first product (p-nitrophenolate), followed by the slower reaction of acetate ion (the other product) with a catalytic lysine residue on the enzyme

b)	the unusual properties of aromatic esters and thus not applicable to the normal chymotrypsin mechanism, which involves the hydrolysis of peptide bonds

c)	the rapid release of p-nitrophenol, followed by the slower formation of the p-nitrophenolate ion

d)	the rapid release of the first product (p-nitrophenolate), followed by the slower hydrolysis of the acyl-enzyme intermediate

Answer 1

Proteases cleave proteins by a hydrolysis reaction—the addition of a molecule of water to a peptide bond.Although the hydrolysis of peptide bonds is thermodynamically favored, such hydrolysis reactions are extremely slow,

(B)chymotrypsin, cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine.

(A) the first reaction is fast and after that the reaction is slow because the saturation stage achieved,

(c) and (D) Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated