Disulfides carboxypeptidase

The shape of a large protein is influenced by many factors including of course Its primary and secondary structure The disulfide bond shown m Figure 27 18 links Cys 138 of carboxypeptidase A to Cys 161 and contributes to the tertiary structure Car boxypeptidase A contains a Zn " ion which is essential to the catalytic activity of the enzyme and its presence influences the tertiary structure The Zn ion lies near the cen ter of the enzyme where it is coordinated to the imidazole nitrogens of two histidine residues (His 69 His 196) and to the carboxylate side chain of Glu 72... 

Human proinsulin has been synthesized in homogeneous solution from 11 protected fragments using azide coupling.1151 The difficulties with insoluble intermediates were sufficiently overcome to allow the 86-residue peptide to be synthesized. The product was de-protected, converted into the 5-sulfonate, and then reduced and reoxidized to form the three disulfide bonds. The product was extensively purified and analyzed, and shown to be pure proinsulin. This product could then be converted into insulin by the use of endopeptidases I and II from pancreatic (3-cell granules, together with carboxypeptidase H, which removed the four basic residues 31, 32, 64, and 65, and split out C-peptide.1 6 ... 

Processing of insulin. Insulin is synthesized by membrane-bound polysomes in the /3 cells of the pancreas. The primary translation product is preproinsulin, which contains a 24-residue signal peptide preceding the 81-residue proinsulin molecule. The signal peptide is removed by signal peptidase, cutting between Ala (—1) and Phe (+1), as the nascent chain is transported into the lumen of the endoplasmic reticulum. Proinsulin folds and two disulfide bonds crosslink the ends of the molecule as shown. Before secretion, a trypsinlike enzyme cleaves after a pair of basic residues 31, 32 and 59, 60 then a carboxypeptidase B-like enzyme removes these basic residues to generate the mature form of insulin. 

Chymotrypsinogen, a single polypeptide chain of 245 amino acid residues, is converted to a-chymotrypsin, which has three polypeptide chains linked by two of the five disulfide bonds present in the primary structure of chymotrypsinogen. tt- and S-chymotrypsin also have proteolytic activity. In contrast, the conversion of procarboxypeptidase to carboxypeptidase involves the hydrolytic removal of a single amino acid. 

The shape of a large protein is influenced by many factors, including, of course, its primary and secondary structure. The disulfide bond shown in Figure 27.18 links Cys-138 of carboxypeptidase A to Cys-161 and contributes to the tertiary structure. Car-... 

The primary structure of a protein is the sequence of its amino acids and the location of all its disulfide bridges. The N-terminal amino acid of a peptide or protein can be determined with Edman s reagent. The C-terminal amino acid can be identified with carboxypeptidase. Partial hydrolysis hydrolyzes only some of the peptide bonds. An exopeptidase catalyzes the hydrolysis of a peptide bond at the end of a peptide chain. An endopeptidase catalyzes the hydrolysis of a peptide bond that is not at the end of a peptide chain. 

The primary structure of a protein is the sequence of its amino acids and the location of all its disulfide bridges. The N-terminal amino acid can be determined with Edman s reagent. The C-terminal amino acid can be identified with a carboxypeptidase. 

Dhariwal 6e McCann, unpublished data, 1966). This observation indicates that intact peptide bonds are required for their activity. Carboxypeptidase failed to inactivate the FRF in one experiment. Thioglycollate splits the disulfide bridge in oxytocin and vasopressin, thus inactivating the molecules, but this treatment is without influence on LRF or hypothalamic CRF (16). Consequently, it appears likely that the chemical structures of LRF and CRF, at least with respect to the disulfide bridge, are dissimilar from that of the known neuroh3q)ophysial pol3q)eptides. 

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