Question

1. What are the three general mechanisms of Catalysis?

Draw the mechanisms of all three of them out, using chymotrypsin as a model enzyme.

Answer 1

Answer-:

Draw the mechanisms of all three of them out, using chymotrypsin as a model enzyme.

Explanation-:

Chymotrypsin -: It is a digestive enzyme belonging to the super family of enzymes are also called serine proteases. It uses in an active serine residue to perform hydrolysis on the C-terminus of the aromatic amino acids of another proteins. Chymotrypsin is a protease enzyme ;that are cleaves on the C-terminal phenylalanine (F), tryptophan (W), and tyrosine (Y) on peptide chains. It is show specificity for aromatic amino acids because its hydrophobic pocket.

Introduction-:Chymotrypsin is a one of the most common studied enzymes due to its two phase kinetics:

1-pre-steady-state and

2-steady state.

The study of these two kinetic states gives evidence of the "Ping-Pong" mechanism, the formation of covalent complexes leading to the covalent hydrolysis reactions, and the rate of the catalyzed reactions. Synthesis of chymotrypsin occurs primarily in the pancreas. Instead of the active form, however, it is produced as an inactive zymogen are called chymotrypsinogen to prevent their  protease activity from digesting the pancreas. The  secretion into the lumen of the small intestine, it is converted to its active form by another enzyme called trypsin. This dependence of the different enzyme for the activation of the protease is a common way for the body to prevent the digestion of organs and other harmful enzymatic side-effects.

1. The target are enters into the active site of chymotrypsin, and it is held by hydrophobic interactions between exposed non-polar groups of enzyme residues and the non-polar aromatic side-chain of the substrate. It is important to note the hydrogen bond between the Schiff nitrogen on histidine-57 and the oxygen side-chain of serine-195.

2. Aided by the histidine-serine hydrogen bonding, the hydroxyl group on serine-195 performs a nucleophilic attack on the carbonyl carbon of an aromatic amino acid while simultaneously transferring the hydroxyl hydrogen to the histidine Schiff nitrogen. This attack pushes the pi carbonyl electrons onto the carbonyl oxygen, forming a short-lived intermediate consisting of a c-terminal carbon with four single bonds: an oxygen anion, the beta-carbon of the aromatic amino acid, the n-terminus of the subsequent amino acid of the substrate protein, and the serine-195 side-chain oxygen.

3. This intermediate is short-lived, as the oxyanion electrons reform the pi bond with the c-terminus of the aromatic amino acid. The bond between the carboxy-terminus of the aromatic amino acid and the n-terminus of the subsequent residue is cleaved, and its electrons are used to extract the hydrogen of the protonated Schiff nitrogen on histidine-57. The bonds between the carbonyl carbon and the serine-195 oxygen remain in an ester configuration. This is called the acyl-enzyme intermediate. The c-terminal side of the polypeptide is free to dissociate from the active site of the enzyme.

4. Water molecules are now able to enter and bind to active site through hydrogen bonding between the hydrogen atoms of water and the histidine-57 Schiff nitrogen.

5. The water oxygen now makes a nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate, pushing the carbonyl’s pi electrons onto the carbonyl carbon as histidine-57 extracts one proton from water. This forms another quaternary carbon covalently bonded with serine, a hydroxyl, an oxyanion, and the aromatic amino acid. The proton on the recently protonated histidine-57 is now able to make a hydrogen bond with the serine oxygen.

6. The oxyanion electrons reform the carbonyl pi bond, cleaving the bond between the carbonyl carbon and the serine hydroxyl. The electrons in this bond are used by the serine oxygen to deprotonate the histidine Schiff nitrogen and reform the original enzyme. The substrate no longer has affinity for the active site, and it soon dissociates from the complex.

Experiments were conducted in 1953 by B.S. Hartley and B.A. Kilby to investigate the kinetics of chymotrypsin-catalyzed hydrolysis. Instead of using a poly-peptide chain as a substrate, they used a nitro-phenyl ester, p-nitrophenyl acetate, that resembles an aromatic amino acid. Hydrolysis of this compound by chymotrypsin at the carbonyl group yields acetate and nitrophenolate, the latter of which absorbs near 400 nm light and its concentration can thus be measured by spectrophotometry

Spectrophotometric analysis of chymotrypsin acting on nitrophenylacetate showed that nitrophenolate was produced at a rate independent of substrate concentration, proving that the only factor contributing to the rate of product formation is the concentration of enzyme; this is typical for enzyme-substrate kinetics. However, when the slope of the 0-order absorbance plot was traced back to the starting point (time = 0), it was found that the initial concentration of nitrophenolate was not 0. In fact, it showed a 1:1 stoichiometric ratio with the amount of chymotrypsin used in the assay. This can only be explained by the fact that hydrolysis by chymotrypsin is biphasic in nature, meaning that it proceeds in two distinct steps.

1. The first step, which describes the initial burst of nitrophenolate seen in Hartley and Kilby’s absorbance plot, is the fastest. The attack of the nitrophenyl acetate substrate by chymotrypsin immediately cleaves the nitrophenolate moiety and leaves the acetate group attached to chymotrypsin, rendering the enzyme inactive.
2. The second step has been deduced to involve the hydrolysis of the acetate group from the inactivated chymotrypsin to regenerate the original enzyme.

Reference-:Chemistry LibreTexts inspark ,www.libretexts.org.