- Proteolysis of dietary protein begins in stomach through the action of pepsin, protease, non-specific hydrolysis
- Pepsin - active enzyme
> Originally synthesized as an inactive zymogen, pepsinogen

> Hydrolyzes proteins in a random, non-specific manner, that is not linked to specific protein sequence

- Pepsinogen - inactive zymogen
> zymogen is defined as an inactive substance which is converted into an enzyme when activated by another enzyme

> Extra 44 amino acids blocks the active site
> Must be clipped away -> when it hits the low pH in the stomach -> unfolds (autoproteolysis) 

> Synthesized and released into stomach

> Activated by low pH in the stomach

- Low pH breaks salt bridges to make this extra piece vulnerable to proteolysis
- Extra 44 amino acids become vulnerable to pepsinogen
- Pepsinogen undergoes autoproteolysis
- Benefits of low pH - activation of pepsinogen and denaturation of dietary proteins to random coils, which are more accessible to protease
- pH 7.3 -> pH 2
- Aspartate is protonated
> There is aspartate in the active site that are responsible for catalytic activity of pepsin

> No longer binds to lysine

- Salt bridge between lysine and aspartate is broken, protein unfolds enough for the inhibitory section of the zymogen to be vulnerable to proteolysis
- Pepsin is a random cleaver - no AA sequence it favors


Protein Breakdown in the Stomach

- Once digestion reaches the duodenum, there is bile coming in alongside pancreatic juices
- Bile contains bicarbonate, which raises pH of food in the gut
- The duodenum is a different place, first part of the small intestine
- Pancreatic juices also have proteolyses - they are activated once they get to the duodenum
- Bile contains bicarbonate and raises pH of food in the gut
~~~~~~~~
Zymogen -> Active Enzyme
- Chymotrypsinogen -> Chymotrypsin
- Trypsinogen -> Trypsin
- Procarboxypeptidase ->Carboxypeptidase
- Proelastase -> Elastase
~~~~~~~~
- Pepsin becomes deactivated - does not do well in a high pH environment
- Good for proteases from pancreas - won’t be randomly cleaved
- Once the pancreatic zymogens get to the small intestine, they becomes proteolysed
- The pancreatic zymogens have left the duodenum
- Enzyme: Enteropeptidase
- Found on surface of small intestine cells
- Transmembrane protein - activity extends into lumen of small intestine
- Zymogens become active enzymes (come from pancreas)
- Trypsinogen encounters enteropeptidase, which cleaves off inhibitory portions of trypsinogen to make it into trypsin
- This works like a cascade, as zymogens become activated they activate surrounding zymogens
- Trypsin will activate all the zymogen that come in from the pancreas
- Why is it stored as inactive? Because the active trypsin would damage the pancreas, where trypsinogen comes from
- Possesses proteolytic activity to activate itself -> *trypsinogen autoactivation*

- These proteolyses are highly specific and cleave at specific amino acid sequences
Active site specificity
> Ex. Tryptin has an Asp in its active site -> specific for positive amino acids -> cleaves after large hydrophobic amino acids
- Positive side chains likes negative side chains (Arginine likes Asp)
- Hydrophobic side chains (Val) will not cleave after a positive side chain
- Trypsin: has a negatively charged amino acid, aspartate, in its active site. 
- Cleaves after positively charged amino acids like lysine and arginine. 
- Chymotrypsin: has a large active site without any bulky hydrophobic residues, without any charged residues
- Cleaves after large hydrophobic amino acids like phenylalanine or tyrosine, or even tryptophan. 
- Elastase: typically cleaves after an alanine or valine. 
- Once the proteins are cleaved, they are taken up by the epithelial cells on the surface of the small intestine into the enterocytes
- Epithelial cells separate the inside of the body from the external environment
- Digestive tract is external environment
- Broken down even more by internal peptidases
- Shuttled in the bloodstream as amino acids


Protein Breakdown in the Duodenum and the Small Intestine

Michigan State University

Protein Degradation

Dietary Protein Breakdown

- primarily in proteosomes and lysosomes
- catabolism of cellular protein
- Reasons
- Regulation through turnover
- Quality control: errors, mistranslated, misfolded
> Chaperons make an attempt at correction, but lysosome and proteasomes degrade

- Source of amino acids: fuel and anabolic metabolites
> Use proteins for fuel and anabolic reactions

- Can also use proteins as anabolic precursors
- Some N-terminal sequences can affect protein longevity
- Serine is long (>20 hours), aspartate very short (< 3 minutes)
- PEST sequence has short halflife
> Proline (P), Glutamate (E), Serine (S), Threonine (T)
> May be sites for phosphorylation that allows its recognition by ubiquitination machinery 
- Make sure we degrade the right protein at the right time
STEPS
- Ubiquitination
- Transamination


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