Lecture 1 pt. II

serine proteases include

trypsin, chymotrypsin, and elastase (digestive enzymes made by the pancreas and are secreted into the intestine to break down proteins)

• hydrolyze peptide bonds and involve a serine residue in the catalysis

• mechanism is both acid/base and covalent catalysis

serine proteases that hydrolyze peptide bonds have

similar tertiary structure and arrangement with loops, but the primary sequences are different

• amino acid residues in the active site are conserved in the S1 binding pocket

the active sites of serine proteases share

common structural components

• catalytic residues of the active site (catalytic triad for chymotrypsin): Ser195, His57, and Asp102

  • essential for the activity of the serine protease

  • the hydroxyl group of Ser195 attacks the carbonyl group of the substrate, and the O- is stabilized by the oxyanion hole by the NHs of Gly193 and Ser195

binding pocket is the

specificity pocket that determines which peptide bonds will be broken and binds to specific amino acid residues on the substrate in a specific and proper orientation

• the catalytic amino acids in the active site can then break peptide bonds on the substrate adjacent to the amino acids sitting in the binding pocket

• binding pocket specificity determines the specific serine protease

• polar amino acids catalyze reactions in active sites

substrate specificity is determined by the

amino acids in the serine protease binding pocket

• different peptide bonds are also oriented in the active site for cleavage

chymotrypsin

large substrate binding pocket accommodates aromatic residues such as tyrosine (cleaves peptide bonds after hydrophobic rings enter)

trypsin

binding pocket with a negatively charged residue at the bottom accommodates residues with a positive charge such as arginine (cleaves peptide bonds after charged residues enter)

elastase

Thr and Val residues close off the binding pocket so that only small residues such as alanine can be accommodated (cleaves peptide bonds after small residues enter)

serine proteases have

three conserved residues that are the catalytic triad

• Ser, His, Asp

  • positions vary based on the serine protease, but the order and arrangement are the same

aspartate and histidine work together to make

serine a highly reactive nucleophile to attack the peptide bond

• the pKa on serine is too high for the deprotonated form to be present in significant concentrations at physiological pH, since they do not lose H+ at physiological pH

• Ser is then linked to His and Asp in a hydrogen bonding network

  • compression of the hydrogen bond between Asp and His occurs when a substrate binds and results in a stronger interaction

  • this shifts the e- density away from the Asp residue and shifts the pKa of His to make it higher (6 to 12) to make it a stronger base

  • His then removes the H+ from the neighboring Ser and deprotonation of Ser makes it a strong nucleophile with a negative charge on O- (oxyanion)

the oxyanion hole is essential for

stabilizing intermediates

• formed from the backbone amino groups of two residues

• negatively charged O- in the hole forms hydrogen bonds with H atoms in the oxyanion hole to stabilize the intermediate

  • during the reaction of chymotrypsin, two short-lived tetrahedral intermediates are formed that contain an O- atom and is representative of the transition state which is a highly energetic state that the reactant must pass through to make the product

• aromatic rings can bind in the hydrophobic pocket, and the substrate tetrahedral intermediate with the oxyanion can be oriented with the Ser and Gly residue

the chymotrypsin mechanism starts with the

substrate binding at the active site

• the enzyme active site contains the substrate specificity pocket, catalytic triad, and oxyanion hole with backbone amino groups of Gly and Ser that stabilize the substrate and keep it in the binding pocket and interacting with the oxyanion O- transiently

1. polypeptide substrate binds to the

enzyme active site

• Phe fits into the substrate specificity pocket, and chymotrypsin breaks the peptide bond between residues and the next residue is not specific to the reaction

the substrate then binds to the

active site, and the aromatic R group of Phe binds to the specificity pocket which positions the carbonyl O atom of the peptide backbone into the oxyanion hole, and the peptide bond between the carbonyl and amino group adjacent to the Ser residue is in the active site

• a conformational change occurs after binding which shortens the hydrogen bond between Asp and His

• the bond between the carbonyl and amino groups is then broken

2. His removes H+ from

Ser, which allows a nucleophilic attack from the Ser O- atom on the carbonyl C atom of the peptide

• this forms a bond between the backbone C atom and the O of Ser, which is a covalent bond between the enzyme and substrate (acyl-enzyme intermediate)

once the substrate is bound, an

acyl-enzyme intermediate is formed

• the C=O bond displaces a pair of e- from the double bond to produce an oxyanion (O-), which is stabilized by the oxyanion hole by the backbone amino groups of Gly and Ser via hydrogen or ion-dipole interactions

• the acyl-enzyme intermediate is the first of two unstable tetrahedral intermediates

  • the central C atom of the intermediate has four attachments, which is stabilized by the additional interactions that form between the transition state and the chemical groups found in the active site of the enzyme

the third step of the mechanism is to

cleave the peptide bond and release the first product

3. His donates H+ to the amino group of the

substrate as a proton donor, resulting in peptide bond cleavage, and the carboxyl-terminal fragment is released as the first product

• the pair of e- on the O atom will return to the double bond, so the tetrahedral intermediate resolves itself, and the formation of the new bond causes slight conformational change

  • bond lengthening occurs between Asp and His and causes the pKa of His to return to 6, so that it can donate H+ again as the e- from the C-N bond abstract the H+ from His

• breaking the peptide bond causes the carboxyl terminal end of the peptide bond to be released from the active site

  • the rest of the peptide is still attached with covalent bonds to the active site through the backbone C atom to the Ser O atom

water enters the active site to help form a

second tetrahedral intermediate

4. water enters the active site, and His acts as a

general base and removes H+ from water, and the resulting OH- acts as a nucleophile and attacks the carbonyl C atom of the backbone of the covalent acyl-enzyme intermediate

• attaches the hydroxyl group from water to the C atom of the substrate backbone to form a second tetrahedral intermediate, where the C atom has four attachments, and a new one to the OH- group

• the oxyanion is stabilized in the oxyanion hole made by the backbone amino groups of Gly and Ser

in the final steps of the mechanism, the product is

released and the active site returns to its original state

5. His donates H+ to Ser, resulting in the

cleavage of the acyl-enzyme intermediate by the C-O bond, and the amino-terminal fragment is released as the second product, and the catalytic triad in the active site is regenerated

• the e- from the O atom return to make the C=O double bond, and the bond between the Ser C atom and O is broken as it abstracts the H+ from His

  • this releases the amino-terminal fragment of the substrate from the enzyme

6. the functional catalytic triad is

regenerated within the enzyme active site

four main points about the serine protease mechanism

1. the position of the catalytic amino acid triad in the active site creates the nucleophile that can cleave the peptide bond (Ser)

2. the nucleophilic attack by Ser creates a transient covalent acyl enzyme intermediate which is an example of covalent catalysis

3. the position of Gly and Ser create a microenvironment called the oxyanion hole which stabilizes an otherwise unstable tetrahedral intermediate

4. His serves as a general acid/base catalyst throughout the mechanism, by abstracting H+ to increase the nucleophilicity of Ser, and then donating H+ to resolve the tetrahedral intermediates