Human pepsin 3b

Marko Krtinic '24 and Catriona Macintosh '23

Contents:

I. Introduction


We are interested in the human protein pepsin as we are keen to discover how it functions in digestion. Pepsin enzymes are endopeptidase proteins characterized by the activity of a peptide hydrolase. Hydrolases catalyse the cleavage of a covalent bond using water. Human pepsin 3b is one of the aspartic proteinase digestive enzymes of mammals (Bailey et al. 2012). It is secreted by gastric chief cells of the stomach lining its inactive form called pepsinogen. The low pH environment from the hydrochloric acid activates pepsinogen to convert into the mature pepsin form. Then in its active form, pepsin functions to break down proteins into smaller peptides during food digestion (Andreeva and Rumsh, 2001).

Human pepsin A has three chromatographically distinct isoforms 1, 3a, 3b and 3c. All of these variants are encoded by the same gene and have identical poly-peptide sequences, differing only in their post transcriptional modifications with pepsin 3b being the major variant (Jones et al. 1995). 


 Human pepsin 3b contains two unique types of molecules; the protein Pepsin A and . Pepsin is bilobal, composed of two almost identical N and C domains related by an intra dyad (Abad-Zapatero et al., 1990). 

II. Primary Structure

The protein pepsin A itself is a monomer composed of 326 amino acid residues, forming the two similar lobes. The is composed of residues 1-170 and the lobe comprises residues 171-325. A significant fraction of the amino acids are polar and buried.

III. Secondary Structure

Several elements of secondary structure are found in pepsin, which are vital in achieving enzymatic function. are the most predominant structure of the domains, accounting for 44% of residues. Additionally, there are ten small right handed which contribute 14% of total residues and several random coils (Harel et al., 2020).

IV. Tertiary Structure

Pepsin has a bilobal tertiary structure as it contains two domains, the (starting with Valine 1) and (ending with Alanine 326). These similar domains come into close proximity upon protein folding and are critical for active site formation and the overall protein function. Pepsin forms three between the sulfur atoms of cysteine residues in the peptide chain which hold important roles in the folding of the protein and stabilizing the two domains (Nakagawa et al., 1971).

V. Active Site

Each lobe contains an active site aspartate residue (Asp-32 and Asp-215) which are held together by an extensive network of (James & Sielecki, 1983). A portion of the target protein substrate approximately 8 amino-acids long is harbored in this catalytic center and pepsin uses the pair of aspartate residues to perform the protein cleavage reaction with water (Harel et al., 2020). The polar nature of the aspartate residues allows hydrogen bonding with respect to both the water and substrate, conferring rigidity and aiding in the correct alignment for the reaction mechanisms of pepsin (Figure 1).


Interestingly, during the pepsin hydrolysis reaction of the peptide bond, the two aspartic residues simultaneously act as both an acid and a base. At the beginning of the reaction, in the low pH environment of the stomach, Asp32 is deprotonated while Asp215 still has its proton (Figure 1a). During the first step there is a nucleophilic attack of the carbonyl carbon of the substrate by water with Asp32 accepting a protein from the water while Asp donates a proton to the substrate. As a result, an intermediate compound called an amide dehydrate is formed. This intermediate then goes onto cleave the peptide bond with the Asp32 now donating a proton to the intermediate as the Asp215 accepts one (Figure 1.c). This action is the step that cleaves the peptide bond in the polypeptide chain hence degrading the protein in digestion. Not shown in the diagram is the last step of the mechanism, in which a proton transfers back from Asp215 to Asp32 reaching equilibrium again so that the reaction can start all over again (Garret and Grisham, 2012).


Figure 1: The reaction mechanisms for pepsin


VI. References

Andreeva, N. S., & Rumsh, L. D. (2001). Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes. Protein Science, 10(12), 2439-2450. https://doi.org/10.1110/ps.ps.25801 


Abad-Zapatero, C., Rydel, T.J. and Erickson, J. (1990), Revised 2.3 A structure of porcine pepsin: Evidence for a flexible subdomain. Proteins, 8: 62-81. https://doi.org/10.1002/prot.340080109 


Bailey, D., Carpenter, E. P., Coker, A., Coker, S., Read, J., Jones, A. T., Erskine, P., Aguilar, C. F., Badasso, M., Toldo, L., Rippmann, F., Sanz-Aparicio, J., Albert, A., Blundell, T. L., Roberts, N. B., Wood, S. P. & Cooper, J. B. (2012). An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases. Acta Cryst, D68, 541-552.

https://doi.org/10.1107/S0907444912004817


Fruton J. S. (2002). A history of pepsin and related enzymes. The Quarterly review of biology, 77(2), 127-147. https://doi.org/10.1086/340729 


Garrett, R. H., & Grisham, C. M. (2012). Biochemistry (2nd ed.). Cengage Learning.

Harel, M., Canner, D., Sussman, J.L., Berchansky, A., Prilusky, J. (2020, October 11). Pepsin. Proteopedia, Life in 3D. https://proteopedia.org/wiki/index.php/Pepsin#What_are_the_Structures_Implications.3F 


Jones, A. T., Keen, J. N., & Roberts, N. B. (1993). Human pepsin 3b peptide map sequence 

analysis, genotype and hydrophobic nature. Journal of chromatography, 646(1), 207-212. https://doi.org/10.1016/s0021-9673(99)87022-8 


Nakagawa, Y., & Perlmann, G. E. (1971). Disulfide bonds of pepsinogen and pepsin: Identification of the disulfide bonds which can be reversibly reduced and reoxidized. Archives of Biochemistry and Biophysics, 144(1), 59-65. https://doi.org/10.1016/0003-9861(71)90454-1 

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