Protein Turnover – Protein and Amino Acids in
2.1 Nitrogen b alance and p rotein r equirements
2.1.1 Protein d igestion and a bsorption
The fi rst stage in protein digestion is denaturation; folded native proteins are relatively resistant to enzymic hydrolysis. Denaturation is achieved by heat, as when foods are cooked, and also by the strong acid of gastric juice. Table 2.2 shows the major human proteolytic enzymes, as well as a number of plant,
Table 2.2 Proteolytic enzymes.
EC number Source Specifi city
Digestive endopeptidases
chymosin (rennin) 3.4.33.4 neonatal gastric juice hydrophobic, preferably aromatic; Phe 105 - Met in κ - casein
pepsin A 3.4.23.2 gastric juice hydrophobic, preferably aromatic
pepsin B (parapepsin)
3.4.23.3 gastric juice hydrolyzes gelatine gastricsin (pepsin
C)
3.4.23.4 gastric juice Tyr - trypsin 3.4.21.4 pancreatic juice Arg - , Lys -
chymotrypsin 3.4.21.1 pancreatic juice Tyr - , Trp - , Phe - , Leu - elastase 3.4.21.36 pancreatic juice Ala -
pancreatic elastase II
3.4.21.71 pancreatic juice Leu - , Met - , Phe - enteropeptidase 3.4.21.9 crypts of Lieberk ü hn Lys 6 - Ile in trypsinogen
Digestive exopeptidases
aminopeptidase 3.4.11.x crypts of Lieberk ü hn N - terminal except Arg or Lys.
carboxypeptidase A
3.4.16.x pancreatic juice C - terminal except - Asp, - Glu, - Arg, - Lys, - Pro carboxypeptidase
B
3.4.16.x pancreatic juice C - terminal Lys or Arg dipeptidases 3.4.13.x mucosal brush
border
various tripeptide
aminopeptidase
3.4.14.x mucosal brush border
N - terminal of a tripeptide
Plant, bacterial and fungal proteases
ananain 3.4.22.31,
3.4.22.32
pineapple, Ananas comosus
broad actinidain 3.4.22.14 kiwi fruit, Actinidia
chinensis
broad, preferably hydrophobic caricain 3.4.22.30 pawpaw, Carica
papaya
broad, preferably hydrophobic chymopapain 3.4.22.6 pawpaw, Carica
papaya
broad, preferably hydrophobic clostripain 3.4.22.8 Clostridium
histolyticum
Arg - collagenase 3.4.24.3 Clostridium
histolyticum
- Gly in collagen
cerevisin 3.4.21.48 Saccharomyces
cerevisiae
broad
endopeptidase K 3.4.21.64 Tritirachium album broad, hydrolyzes keratin fi cin (fi cain) 3.4.22.3 fi g, Ficus glabrata broad, preferably
hydrophobic
bacterial and fungal enzymes that are important in food technology and labo-ratory studies; Table 2.3 shows the Enzyme Commission classifi cation of proteolytic enzymes, based on their mechanisms of action.
The fi rst stage of enzymic digestion of proteins occurs in the stomach, cata-lyzed by pepsin. Pepsin is secreted as an inactive zymogen, pepsinogen. This is activated either by acid hydrolysis (which removes the peptide fragment that overlies and blocks the catalytic site in the zymogen) or by enzymic hydrolysis (to remove the blocking peptide, catalyzed by pepsin that has already been activated). Pepsin is an endopeptidase that catalyzes hydrolysis of peptide bonds adjacent to large neutral amino acids (the aromatic and branched- chain amino acids and methionine), so the result of pepsin action is the production of a relatively large number of peptide fragments. Gastric digestion may result in up to half of the ingested protein being hydrolyzed to peptides smaller than ten amino acids.
Table 2.3 The Enzyme Commission ( EC ) classifi cation of peptidases.
3.4.11.x aminopeptidases 3.4.13.x dipeptidases
3.4.14.x dipeptidyl peptidases and tripeptidyl peptidases 3.4.15.x peptidyl dipeptidases
3.4.16.x serine - type carboxypeptidases (Ser in catalytic site)
3.4.17.x metallocarboxypeptidases (zinc or another divalent ion in the catalytic site) 3.4.18.x cysteine - type carboxypeptidases (Cys in the catalytic site)
3.4.19.x omega peptidases (peptide bonds other than α - carboxyl - α - amino) 3.4.21.x serine endopeptidases (Ser in the catalytic site)
3.4.22.x cysteine endopeptidases (Cys in the catalytic site) 3.4.23.x aspartic endopeptidases (Asp in the catalytic site)
3.4.24.x metalloendopeptidases (zinc or another divalent ion in the catalytic site) 3.4.99.x endopeptidases of (as yet) unknown mechanism
EC number Source Specifi city
fruit bromelain 3.4.22.32 pineapple, Ananas comosus
broad mucorpepsin 3.4.23.23 Mucor pusillus, M.
miehei
hydrophobic, clots milk
oryzin 3.4.21.63 Aspergillus oryzae broad
papain 3.4.22.2 pawpaw, Carica
papaya
broad, preferably hydrophobic stem bromelain 3.4.22.32 pineapple, Ananas
comosus
broad subtilisin 3.4.21.62 Bacillus subtilis broad thermolysin 3.4.24.27 Bacillus
thermoproteolyticus
- Leu, - Phe Table 2.2 Continued
Protein digestion continues in the small intestine, where three more inac-tive precursors of endopeptidases are secreted in the pancreatic juice:
trypsinogen, chymotrypsinogen and pro - elastase. Trypsinogen is activated to trypsin by the enzyme enteropeptidase (sometimes called enterokinase, although it is not a kinase), secreted by the crypts of Lieberk ü hn in the duo-denum. Once activated, trypsin catalyzes the activation of chymotrypsinogen to chymotrypsin, and of pro - elastase to elastase. Like pepsinogen, these three zymogens are inactive because of a terminal peptide region that overlies and blocks the active site, and activation results from cleavage of this blocking peptide.
Trypsin, chymotrypsin and elastase are so - called serine proteases, with a serine residue at the catalytic site. These three enzymes show very consider-able sequence homology, and the catalytic site is made up by histidine 57, aspartate 102 and serine 195. In all three enzymes, the peptide substrate sits in a groove in the surface of the enzyme, with the amino acid that provides the carboxyl group of the bond to be cleaved sitting in a pocket under the groove. This explains the different substrate specifi cities of the enzymes:
• In trypsin, which catalyzes hydrolysis of esters of lysine and arginine, there are two glycine residues on the sides of the substrate - binding pocket and an aspartate residue at the base, so providing a negative charge to attract the positive charge of the substrate.
• In chymotrypsin, which catalyzes hydrolysis of the esters of aromatic amino acids, the amino acid at the base of the pocket is serine.
• In elastase, which catalyzes hydrolysis of esters of small neutral amino acids, the substrate - binding pocket is blocked by a valine and a threonine residue on the sides.
The end result of the actions of these endopeptidases is the liberation of a large number of small peptides that are substrates for aminopeptidases and carboxypeptidases, which catalyze hydrolysis of the amino - and carboxy -terminal amino acids of peptides respectively. Procarboxypeptidases are secreted in the pancreatic juice and pro - aminopeptidases by the crypts of Lieberkü hn, and these are activated by partial proteolysis catalyzed by trypsin. Neither amino - nor carboxypeptidases acts on di - and tripeptides, which are hydrolyzed by di - and tripeptidases in the brush border of intestinal mucosal cells.
As shown in Figure 2.1 , in addition to the dietary intake of protein of some 90 g/day, a considerably larger amount of endogenous protein and other nitrogenous compounds enters the intestinal tract. Most of the protein is
hydrolyzed and the resultant amino acids are absorbed. Much of the endog-enous protein consists of digestive enzymes secreted in to the gastro - intestinal tract and proteins in mucus, which is secreted in order to protect the intestinal mucosa from attack by digestive enzymes, together with shed mucosal cells.
In addition, some of the proteins synthesized by intestinal bacteria are hydro-lyzed, and the resultant amino acids are absorbed in the large intestine. Much of the faecal loss of protein is mucin, which is very resistant to enzymic hydrolysis (Bergen & Wu, 2009 ; Fuller & Reeds, 1998 ).
As will be discussed in section 2.5 , amino acids are absorbed from the intestine by a number of different transport proteins, depending on the chemistry of their side chains. In most cases, amino acid uptake is by sodium dependent secondary active transport. Di - and tripeptides are taken up into intestinal mucosal cells by a separate set of transporters from those that are involved in the absorption of free amino acids, and are hydrolyzed intracellularly.
There is also signifi cant absorption of intact proteins and relatively large peptides from the intestinal lumen. This is especially important in neonates, who acquire passive immunity from the maternal antibodies in milk (espe-cially colostrum), but it also continues throughout life. The uptake of intact proteins and peptides may be either paracellular, by diffusion through the
Figure 2.1 Nitrogen balance – protein fl ux through the gastro - intestinal tract.
dietary protein 90 g
faecal loss (10 g)
enzymes intestinal cells
mucus
amino acids dipeptides
body protein
~ 10 kg
amino acids
metabolites
urine excretion (80 g) 200 g
280 g
tight junctions between epithelial cells, or transcellular, by endocytosis into the intestinal M cells in Peyer ’ s patches, followed by transfer of (potential) antigens to sub - epithelial lymphocytes. Such absorption of intact proteins and relatively large peptides is a major factor in food allergy.
In addition, a number of smaller peptides are also absorbed intact. Many of these resemble the neuro - active endorphins and, since they have some endorphin- like activity, they have been termed exorphins (Gardner, 1988 ).