The DPP-IV active site exists at the intersection of a α/β hydrolase domain and a β-propeller domain and consists of the catalytic triad and supporting residues. The catalytic triad is made up of a nucleophile, Serine630, a base, Histidine740, and an acid, Aspartate708. Also essential to the cleaving mechanism are two supporting residues, Tyr547 and Tyr631, that stabilize the oxyanion tetrahedral intermediate by forming hydrogen bonds with the oxyanion. In addition, there are anchoring residues that coordinate the carbonyl of the N-terminal amino acid residue of the substrate and align it for the nucleophilic attack by Ser630. These residues are Glu205, Glu206, Asn710, and Arg125.
Animation of catalytic triad and supporting residues in DPP-IV active site:
Creation of Nucleophile
The hydrogen bond between the carboxyl group of aspartate, which is deprotonated at physiological pH, and histidine increases the pKa of the imidazole nitrogen of histidine from 7 to 12, thus increasing its basicity. It also aligns histidine within the active site by restricting rotation of its side chain.
The imidazole nitrogen of the histidine accepts the hydrogen from the alcohol group of the serine.
The pKa of the hydrogen of serine is reduced by the positively charged histidine positioned next to it. This increases the nucleophilicity of serine.
Mechanism of Cleavage
The substrate binds to the surface of DPP-IV such that the peptide bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond positioned near the serine.
Serine’s hydroxyl group attacks the carbonyl carbon of the peptide, and the pair of electrons forming the double bond to the peptide’s carbonyl oxygen moves to the oxygen, resulting in a oxyanion tetrahedral intermediate.
Oxyanion hole: The alchohol group of Tyr547 and the backbone -NH of Tyr631 form hydrogen bonds with the oxyanion intermediate, stabilizing its negative charge.
The peptide bond is now broken. The covalent electrons creating this bond attack the hydrogen of histidine. The electrons that previously moved from the carbonyl oxygen double bond to create the oxyanion move back to recreate the bond, generating an acyl-enzyme intermediate.
Water replaces the N-terminus of the cleaved peptide and attacks the carbonyl carbon. The electrons from the double bond move to create another oxyanion, and a bond between the oxygen of the water and the carbon is formed. This is coordinated by the nitrogen of the histidine, which accepts a proton from the water. Another tetrahedral intermediate is generated.
The electrons of the bond formed in the first step between the serine and the carbonyl carbon now attack the hydrogen that histidine had just bound. The double bond is reformed again between the now electron-deficient carbonyl carbon and the oxygen.
The C-terminus of the peptide is ejected.
Supporting Residues
There are two supporting, Tyr547 and Tyr631, residues that are essential to the stabilization of the oxyanion tetrahedral intermediate. By forming hydrogen bonds with the oxyanion they create the oxyanion hole.
There are also anchoring residues, Glu205, Glu206, Asn710 and Arg125, that coordinate the carbonyl of the N-terminal amino acid residue of the substrate and align it for the nucleophilic attack by Ser630.
Inhibition
Vildagliptin:
The mechanism that creates the nucleophilic serine is also essential for inhibitor binding.
The nucleophilic -OH of Ser630 attacks the nitrile carbon of Vildagliptin instead of carbonyl carbon of the peptide, resulting in the formation of an imidate adduct.
This results in inhibition of DPP-IV via reversible covalent binding.
Chymotrypsin
Chymotrypsin is another serine protease that acts by the same digestive mechanism as DPP-IV. It is found in the intestines of many organisms.
Authors
Jessica Pakonen, Kaylyn Billmeyer, Daniel Balle, McKinley Shifflett
St. Olaf College.
Medicinal Chemistry, Interim 2018.
Professor Robert Hanson.
