Term Paper on Protein Digestion | Biomolecules | Biology

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The proteolytic enzymes of the digestive tract are of two kinds; the endopeptidases, which attack polypeptide chains at peptide bonds away from the ends, and the exopeptidases, which attack the terminal peptide bonds. Proteins are much more readily attacked if they have first been denatured by cooking or the action of gastric HCl.

Endopeptidases:

Pepsin is secreted in the gastric juice as an inactive precursor, pepsinogen. Pepsinogen (mol. wt 42500) is converted into a pepsin-inhibitor complex and various small peptides spontaneously at pHs below 6.0. The reaction is very slow at pH 6.0 but almost instantaneous at pH 2.0. The reaction is auto-catalysed by pepsin. At pHs below 5.4 the inhibitor (mol. wt 3100) dissociates from pepsin (mol. wt 34500), but the complex will re-form at pHs above 5.4. Both pepsin and the inhibitor are hydrolysed to peptides by pepsin itself.

Pepsin is a very acidic protein with an isoelectric point less than pH 1 and an optimum pH of 1.5 – 2.5 depending on the substrate. It is stable in acid solution but is rapidly inactivated in neutral or alkaline solutions; it has no prosthetic group. When acting on synthetic substrates it attacks peptide bonds between an acidic amino acid and an aromatic one ((Asp or Glu)-(Tyr or Phe)).

 

From its action on insulin, however, it is known to attack, among others, links between two aromatic amino acids (Phe-Phe or Phe-Tyr) and links adjacent to leucine (Leu-Val and Tyr-Leu). Pepsin, like rennin, will coagulate milk by converting the phospho-protein caseinogen to casein which forms an insoluble complex with calcium.

The gastric HCl is secreted by the oxyntic cells of the gastric mucosa; at the same time the blood coming from the mucosa is made more alkaline. The cells contain carbonic anhydrase whose presence appears necessary for HCl secretion; about 80 % of the H+ secretion into gastric juice may fail to appear in the presence of the carbonic anhydrase inhibitor acetazolamide (Diamox).

The juice then contains much more Na⁺ ion than usual. It is probable that the hydrogen ion, secreted together with chloride, is derived from water and the remaining hydroxide ion reacts with CO₂ under the influence of carbonic anhydrase to form bicarbonate. The H⁺ almost certainly traverses the membrane by means of a proton pump; whether there is a specific CI^– pump as well is not known.

It is very doubtful whether non-ruminant gastric juice contains rennin, an endopeptidase with a particularly powerful milk-clotting action. It has been reported that human milk is not clotted by calf rennin.

Trypsin is secreted by the pancreas as an inactive precursor, trypsinogen, which is activated by the enzyme enterokinase, secreted by the intestinal mucosa, and then auto-catalytically by trypsin itself. A hexapeptide, Val • (Asp)₄ • Lys, is split off the N-terminal end of trypsinogen during activation by trypsin, leaving an N-terminal isoleucine.

Trypsin has no prosthetic group; it is relatively stable to heat in acid solution but less so in alkaline solution. The optimum pH is in the range 7-9 and it has a low Michaelis constant, indicating that the substrate is firmly bound to the enzyme. It catalyses the hydrolysis of peptides, amides, and esters where a diaminomonocarboxylic amino acid (Lys or Arg) provides the carboxyl group. When acting on a natural substrate it also splits other bonds, e.g. Arg-Gly, Lys-Ala, Phe-Tyr, Lys-Tyr, Arg-Arg, Arg- Ala, and Tyr-Leu.

The chymotrypsin group of enzymes is all derived from a common precursor chymotrypsinogen secreted by the pancreas. The activation is initially brought about by trypsin to give an active chymotrypsin which may be converted to other chymotrypsin by autolysis-there is no change in the molecular weight in the first step, but there are differences in the subsequent products. The optimum pH is 7-8, the Michaelis constant is high, and the enzymes have no prosthetic group. These enzymes attack peptides or esters of a number of amino acids, but particularly non-polar ones (Leu-, Tyr-, Phe-, Met-, Trp-).

Elastase (pancreatopeptidase E), from the pancreas, hydrolyses peptide bonds adjacent to small neutral amino acid residues such as Ala, Gly and Set.

These endopeptidases (pepsin, trypsin, the chymotrypsins, and elastase) bring about the hydrolysis of large protein molecules to smaller peptide fragments. Their further hydrolysis then depends on the action of a number of exopeptidases and dipeptidases either secreted by the pancreas or to be found within the cells lining the intestinal mucosa.

Exopeptidases:

A number of these enzymes, in contrast to the endopeptidases, require a metal ion as activator. The two carboxypeptidases are secreted as precursor pro­carboxypeptidases which are activated by trypsin. Carboxypeptidase A contains firmly bound Zn2+ and hydrolyses off the carboxyterminal amino acid unless this is lysine or arginine. Carboxypeptidase B hydrolyses peptides with carboxy-terminal lysine or arginine. Neither will attack dipeptides.

Leucineaminopeptidase is the best characterized of the intracellular exopeptidases that complete the digestion of protein in the gut. It brings about the hydrolysis of amino-terminal residues from peptides, but not from dipeptides. In spite of the name, it is rather unspecific. There is also a prolidase which catalyses the hydrolysis of proline peptides which are mainly derived from the breakdown of collagen.

Amino Acid Pool:

It is constituted by amino acid present in blood and the extra cellular fluid. Amino acids enter this pool form dietary proteins, after from keto acids (non-essential amino acids) and from tissue protein breakdown. Amino acids from this pool are used for synthesis of tissue proteins and synthesis of certain other important compounds amino acids do not return to the pool. Amino acid over and above synthetic needs are deaminated and degraded.

There is only limited storage of amino acids as proteins in certain tissues. After deamination carbon skeletons of amino acids are used for oxidation (for energy), and for synthesis of glucose and fats.

Protein degradation in the cells is mediated either by the lysosomal proteases (cathepsins) or cytoplasmic protein hydrolysing enzymes. The latter enzymes include proteases acting on protein molecules as endopeptidases and the peptidases which act on small peptides. These peptidases include endopeptidases, amino peptidases and carboxypeptidases.

Extra cellular proteins, membrane associated proteins and intracellular proteins with long half-lives are degraded in lysosomes. Cytoplasmic proteolytic enzymes are important for degradation of abnormal or damaged proteins. Such proteins are targeted for degradation by ubiquitin as explained under “Preparing proteins for degradation”.

Uptake of Amino Acids by Cells from Circulation (g-Glutamyl Cycle):

Reactions of this cycle help transport of amino acids from extracellular fluid into the cells, also probably from intestinal lumen into the mucosal cells the tubular fluid into the renal tubular cells. For operation of this cycle the membrane bound g–glutamyl trans­ferase should be available to react with intracellular glutathione. Possibility of this, however, may not exist in all types of cells.

The plasma membrane bound g-glutamyl transferase transfers the amino acid (under transport) to the g-carboxylate in gluta­thione (GSH) releasing cysteinyl glycine. In the next step g-glutamyl amino acid breaks into 5-oxoproline and the amino acid.

In the remaining part of the reactions there is synthesis of GSH in four steps as shown to start the cycle again.

In this cycle the energy for transport of amino acid into the cell is provided by hydrolysis of peptide bond of GSH. Next, 3ATP as required to synthesize GSH.

g-Glutamyl transferase activity of the serum appears to be derived mostly from hepato biliary system. Serum level of this enzyme is increased in interahepatic and post hepatic biliary obstruction.

In an inborn error there is deficiency of 5-oxoprolinase. In this condition large amount of oxoproline appears in urine.

Preparing Proteins for Degradation:

Many circulatory glycoproteins like immunoglobulins and peptide hormones are removed from circulation for degradation by special mechanisms. Freshly synthesized, their oligosaccharide units bear a terminal sialic acid residue. After hours or days (depending upon half-life) the terminal sialic acid residues of the oligosaccharide chains are removed by asialylases on endothelial cells of blood vessels.

The asialoglycoprotein thus formed is bound on asialoglyco­protein receptors on hepatic cells. After binding the receptor- protein complex is internalized for degradation. A similar mechanism operates for removal of asialoerythrocytes by Kupffer cells of liver.

Proteins which are defective due to inaccurate translation or which are damaged by oxygen free radicals or because of some other reason are prepared for destruction by conjugation with ubiquitin. Ubiquitin is a small protein which is highly conserved (yeast and mammalian ubiquitins differ only in a few amino acids) in evolution.

The carboxylic group of terminal glycine of ubiquitin is covalently linked to e-amino group of lysine residues of protein through a complicated enzymatic process involving three enzymes. Readiness with which, ubiquitin links with a cystsolic protein determines the half-life of the protein.

More readily ubiquitin links, lesser the half-life. Readiness of linking in turn is determined by the amino terminal residue of the protein. Linking is favoured with leucine, phenyl alanine and arginine and linking is prevented with methionine glycine and serine in the amino terminal position.

Daily protein turnover is estimated to be about 400 g. The digestive enzymes, the proteins of sloughed out epithelial cells of gastrointestinal tract and haemoglobin are also important in the turnover process. Half-life of mucosal cells of gastrointestinal cells is 4 to 6 days. Proteins derived from degradation of these cells amount to about 70 g. These are digested and resulting amino acids absorbed.