Pepsin
Created by Megan Gutierrez
Porcine pepsin is a proteolytic enzyme which resides in the stomach of Sus scrofa. Pepsin's role in the stomach is to digest proteins through the hydrolysis of peptide bonds(1). At the optimal pH of 2, pepsin physiologically exists in monomeric form. At higher pH values of 4.5-6, however, aggregates and possible oligomers have been observed(2). These aggregates have yet to be carefully studied and their structures and properties are unknown. Pepsin is thought to exist in different conformations at pH values conducive to aggregate formation, but the structures of these possible alternate conformations are not well characterized(2).
One subunit of 326 amino acids comprises pepsin, which has a molecular weight of 34,509.83g and an isoelectric point of 3.24(3). Pepsin is classified as bilobal, consisting of two domains which become close in proximity upon proper protein folding. The interaction between Asp-32 and Asp-215, each residue located on a different lobe, leads to formation of the active site(4). Pepsin catalyzes the hydrolysis of peptide bonds between hydrophobic or aromatic residues of a protein substrate. Bonds such as Phe-Phe, Phe-Trp, and Phe-Tyr are commonly hydrolyzed(1). The bilobal structure of pepsin is critical to the formation of the active site and thus to the protein's overall function. The hydrolysis reaction carried out by pepsin contributes greatly to digestive capability and nutrient absorption, enabling organisms to obtain molecules essential for survival.
Pepsin contains elements of secondary structure including alpha helices, beta sheets, and random coils. According to sequence data provided by the Protein Data Bank, pepsin is composed of fourteen percent helices and forty-four percent beta sheets. There are ten alpha helices encompassing forty-six residues and thirty-two beta strands encompassing one hundred forty-four residues(5). Random coils are indicated as well. Secondary structure is crucial in achieving enzymatic function. Two antiparallel beta strands on the surface of the protein containing residues Leu-71 through Gly-82 form a loose, functionally important residues. Of paramount importance are Asp-32 and Asp-215, the residues of the Pepsinogen differs from pepsin in that it contains an additional forty-four amino acids on the N-terminus of the chain. These extra amino acids are termed the prosegment(6). In pepsinogen, the prosegment interacts with the other amino acids through electrostatic interactions between acidic and basic residues. At the acidic pH of gastric juice, the acidic residues become protonated and lose their charge, decreasing their affinity for basic amino acids, and pepsinogen is destabilized. An undefined autocatalytic process which involves Thr-77 can then occur. Thr-77 is hypothesized to have a role in the proteolytic cleavage which liberates the prosegment and allows pepsin to achieve its native conformation(9).
A conserved lysine residue has an important function in pepsin. Lys-320 is near the C-terminus of the peptide and is located near both the active site and the flap region of folded pepsin. This positively charged residue aids interacts with the flap to influence flap stability and motility, impacting the speed of catalysis(10).
Finally, the residues His-53 and Gln-55 contribute to ligand interaction. Dimethyl sulphoxide is a polar ligand and forms hydrogen bonds with these residues to destabilize and denature pepsin. Water molecules play a role in this destabilization as well(11). The new interactions change pepsin's secondary structure and lead to loss of function. The purpose of introducing DMSO into a pepsin solution is to study the processes which occur during protein denaturation(11).
DMSO is not the only molecule which can interfere with pepsin's activity. Pepsin is known to be inhibited by pepstatin, which interferes with aspartic proteases. Pepstatin is a small molecule derived from the bacterial species Actinomyces and its mode of inhibition has yet to be discovered. Pepstatin was once used in experimental trials to treat gastric ulcers, but was ultimately unsuccessful(12).
Human uropepsin (PDB ID=1FLH) shares many structural features with pepsin, though their functions vary slightly. A BLAST search was conducted to find a protein with similar primary structure to pepsin. An E-score of 7e-163(13) illustrates that uropepsin and pepsin are similar in primary sequence. The proteins share 86.5% sequence similarity. In addition, both have residues Asp-32 and Asp-215 at the catalytic site and are thus aspartic proteases(14). A Dali search was then conducted to find a protein with similar tertiary structure to pepsin. Results indicated that uropepsin was a match once again based on a Z-score of 52.6 and rmsd=0.6A(15). Pepsin and uropepsin share a bilobal structure of mostly beta sheets(14). These similarities in primary and tertiary structures translate to similarity in overall function. Both proteins function to cleave peptide bonds(16), though uropepsin operates in the bloodstream and has exocrine function while pepsin operates only in the stomach(14). It can be concluded that pepsin and uropepsin have similarities in structure and function.