Polypeptide chain termination hydrolysis

Chymotrypsin-like proteinases are serine proteinases that recognize pqDtide residues with aromatic side chains (phyenylalanyl or tyrosyl residues) and that effect hydrolysis of the polypeptide chain on the carboxy-terminal side of these residues. Examples of chymotrypsin-like proteinases are chymotrypsin and cathepsin-G. 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

Termination Three codons (UAA, UAG and UGA) are stop codons which do not code for any amino acid but, instead of attaching to a tRNA molecule, they bind a protein release factor. When one of these factors is encountered by the ribosome, peptidyl transfer is aborted, the completed polypeptide chain released by hydrolysis and the ribosome subunits separate. The N-terminal methionine unit is then removed from the polypeptide chain. 

The repetitive cycle to identify a sequence of N-terminal amino acids has been automated. In practice, it is limited to about 20-30 amino acids, since impurities build up and the reaction mixture becomes too complex to yield unequivocal results. The usual approach is to break the polypeptide chain into smaller fragments by partial hydrolysis, preferably at positions relating to specific amino acid residues in the peptide chain. There are ways of doing this chemically, and the enzymes chymotrypsin... 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

The start of protein synthesis is signalled by specific codon-anticodon interactions. Termination is also signalled by a codon in the mRNA, although the stop signal is not recognized by tRNA, but by proteins that then trigger the hydrolysis of the completed polypeptide chain from the tRNA. Just how the secondary and tertiary structures of the proteins are achieved is not yet clear, but certainly the mechanism of protein synthesis, which we have outlined here, requires little modification to account for preferential formation of particular conformations. 

The third step is to determine the polypeptide chain end groups. If the polypeptide chains are pure, then only one N-terminal and one C-terminal group should be detected. The amino-terminal amino acid can be identified by reaction with fluorodinitrobenzene (FDNB) (fig. 3.18). Subsequent acid hydrolysis releases a colored dinitrophenol (DNP)-labeled amino-terminal amino acid, which can be identified by its characteristic migration rate on thin-layer chromatography or paper electrophoresis. A more sensitive method of end-group determination involves the use of dan-syl chloride (see Methods of Biochemical Analysis 3B). 

Carboxypeptidase A is a metalloenzyme (containing Zn+ ) which hydrolyzes the C-terminal peptide bond in polypeptide chains (1-4). The hydrolysis occurs most readily when the terminal amino acid residue has an aromatic (or a large aliphatic) R group (cf. 38-> 39+40). 

The role of catalysis in membrane assembly is emphasized again by the above model since the N-terminal sequence of the nascent polypeptide chain of a spanning protein is released by proteolysis as soon as it reaches the cytosol. The N-terminal polypeptide chain extension may help the chain penetrate the hydrophobic bilayer and solubilize the resulting hydrophobic N-terminal part of the chain in the aqueous medium of the cytoplasm. However, the role of the protease-catalyzed hydrolysis of the polypeptide chain in membrane assembly is minimized in the membrane trigger hypothesis (99). According to this model, the essential role of the leader sequence would be to modify, in association with the lipid bilayer, the folding pathway of the protein in such a way that the polypeptide chain could span the membrane. 

When a stop codon appears at the A site translation is terminated. There are no tRNA s that recognize stop codons. Instead releasing factors, eRF, recogiiize the stop codon. The releasing factors along with peptidyl transferases and GTP catalyze the hydrolysis of the bond between the polypeptide chain and the tRNA. The protein and tRNA disassociate from the P site and the ribosome dissociates into the 40S and 60S subunits releasing the mRNA. 

Lactase, which catalyzes the hydrolysis of lactose, is also a bifunctional enzyme. Lactase resides on the same polypeptide chain as phlorizin hydrolase. The entire protein is called lactase-phlorizin hydrolase. The N-terminal end of the polypeptide resides in the lumen, and bears the lactase activity. The central portion of the... 

B. The side chains of the amino acid residues in proteins contain functional groups with different pKs. Therefore, they can donate and accept protons at various pH values and act as buffers over a broad pH spectrum. There is only one N-terminal amino group (pK=9) and one C-terminal carboxyl group (pK= 3) per polypeptide chain. Peptide bonds are not readily hydrolyzed, and such hydrolysis would not provide buffering action. Hydrogen bonds have no buffering capacity. 

The cycle of peptide-chain elongation continues until one of the three stop codons (UAA, UAG, UGA) is reached. There is no aminoacyl-tRNA complementary to these codons, and instead a termination factor or a release factor (RF) with bound GTP binds to the ribosome and induces hydrolysis of both the aminoacyl-linkage and GTP, thereby releasing the completed polypeptide chain from the ribosome. The 475 amino acid-long sequence of rabbit liver RF has been deduced from its cDNA sequence, and it shows 90% homology with mammalian trypto-phanyl-tRNA synthetase (Lee et al., 1990). It has also been reported that for efficient and accurate termination, an additional fourth nucleotide (most commonly an A or a G) after the stop codon is required (Tate and Brown, 1992). The exact role of the fourth nucleotide in the termination of protein synthesis is not fully understood at present. 

The chain elongation process continues and polypeptide synthesis continues until the ribosome complex reaches a stop codon (UAA, UAG, or UGA) on the mRNA. At that point, a specific protein known as a termination factor binds to the stop codon and catalyzes the hydrolysis of the completed polypeptide chain from the final tRNA. The empty ribosome dissociates and can then bind to another strand of mRNA to once again begin the process of protein synthesis. 

The observations that have been summarized immediately suggest structure-function relationship. It seems that a 15-residue segment of the C-terminal polypeptide chain is not essential to activity. Changes in the amino acid sequence can take place without inducing loss of activity. Such a view is supported by degradation experiments. Pepsin degradation of the 39-resi-due polypeptide leads to the formation of three smaller but active polypeptides one of 28, one of 30, and one of 33 amino acid residues. Each of these polypeptides includes the N-terminal serine. In contrast, enzymic (carboxypeptidase, pepsin, chymotrypsin) hydrolysis of the 24 N-terminal polypeptide destroys hormonal activity. Furthermore, the elimination of the single amino acid of the 24 N-terminal peptide seems to inactivate the molecule completely. 

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