Chain of insulin

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

Neutral endopeptidase (NEP, nephrilysin) is an enzyme that preferentially catalyzes cleavage at the amino group of hydrophobic residues of the B-chain of insulin... 

In the synthesis of the A-chain of insulin, 21 amino acids must be combined in a known order. The first steps use a protected dlpeptide (22) and protected cysteine (21)... 

We need a few words about the disulfide bonds that link the two chains of insulin. The side chain of Cys is —CH2—SH. The —SH group is termed the sulfhydryl group. If two of these side chains come together, the sulfhydryl groups can be linked together (oxidized) to form a disulfide bond ... 

This lysosomal endopeptidase [EC 3.4.23.5] is similar to pepsin A, except that the specificity is narrower and will not hydrolyze the Gln" —His peptide bond in the B chain of insulin. The enzyme is a member of the peptidase family Al. 

A few proteins contain two or more polypeptide chains linked covalently. For example, the two polypeptide chains of insulin are linked by disulfide bonds. In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains. 

FIGURE 12-7 Activation of the insulin-receptor Tyr kinase by autophosphorylation. (a) In the inactive form of the Tyr kinase domain (PDB ID 11RK), the activation loop (blue) sits in the active site, and none of the critical Tyr residues (black and red ball-and-stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between Tyr1162 and Asp"32, (b) When insulin binds to the a chains of insulin receptors, the Tyr kinase of each /3 subunit of the dimer phosphorylates three Tyr residues (Tyr"58, Tyr"62, and... 

In 1950, when the study of ribosomes began, no methods for determining the sequences of amino acids in proteins or of nucleotides in nucleic acids existed.11 Sanger published the sequences of the two short chains of insulin in 1953, and the first transfer RNA sequence was published by Holley in 1965.21 Never-... 

Steps involved in the sequence determination of the B chain of insulin. Amino acids are represented here by their single-letter codes (see table 3.1). 

In crystal structures the two chains of insulin form highly ordered globular structures (8). Two main structural types form depending on crystallization conditions. In both structures the A-chains form two CC-helical segments, from residues Al—A8 and A13—A19, which are connected by a turn. In the structure referred to as the T-state, the B-chain contains two regions of extended chain, Bl—B8 and B21—B30, connected by an Ot-helix from B9-B19. In the R-state structure, the B-chain helix extends from Bl—B19. The crystallographic T-state structure best matches the solution structure of insulin determined by nmr (9), although the R-state can be induced in solution under the appropriate conditions. The surface of insulin which interacts with the insulin receptor includes the N- and C-termini of the A-chain and the C-terminus of the B-chain. 

Fig. 27. Space-filling model of the B chain of insulin. Numbers indicate position of carboxyl oxygens (after Warner231)). The MM in nacreous layer of molluscs is assumed to be built according to the same principles...

In biopolymers hydrates may enforce special conformations141. For example, the harmone oxy tocine of the hypophysis conforms with all hydrophobic groups on one side1271 (Fig. 26) and the hydrophilic groups on the other12 (Fig. 27). Similarity the A-chain and the B-chain of insulin can be arranged to form a disc with a... 

A. M. Blow. Action Of human lysosomal elastase on the oxidized B chain of insulin. Buchan. / 7 7 13(1977). 

The procedure for insulin is more complex because insulin is synthesized as preproinsulin and must be processed to yield the A and B chains of insulin (page 415). Bacteria do not have the processing system for converting the precursor to insulin. The appropriate cDNAs for the A and B chains were coupled into individual plasmids and each inserted into separate bacteria for production of the A and B chains. Following purification of the individual chains/ the proper disulfide bonds were chemically formed to yield the mature insulin. 

The two chains of insulin, fragments of what was originally a single chain, are held together by covalent disulfide bonds. Hemoglobin has four polypeptide chains, held together by noncovalent interactions only. 

Fig. 6. Structura, resemblances between the A and B chain of insulin (beef).

It has already been remarked that most enzymes with elastolytic activity have proved to be proteinases with a wide peptide-bond specificity. Thus papain, bromelin, and ficin have a similarly broad hydrolytic action and are all active elastolytic enzymes. Sanger et al. (1955) found that the oxidized A chain of insulin was hydrolyzed in a variety of positions by either crude papain or activated mercuripapain. There were five major... 

With oxidized -chain of insulin, as substrate, except where noted. 

Table VI. Cleavage Sites on S-Sulfo B-Chain of Insulin by Acidic Proteases""...

Table VII. Penicillopepsin Nature of Amino Acids at Cleavage Points in Glucagon and B-Chain of Insulin ...

In an attempt to find out more about the nature of the secondary binding site in penicillopepsin. Mains et al. (76) analysed the nature of the amino acid side chains at positions removed from the sensitive peptide bond. An abbreviated summary of the results is given in Table VII. The number of hydrophobic and hydrophilic side chains respectively are listed for four amino acids on either side of every peptide bond broken in the B-chain of insulin and in glucagon. The positions are numbered Pi, P2, P/, P2 etc. as defined by Berger and Schechter (103). The choice of four positions was taken from the Frutons work (73) on the specificity of pepsin and the eflFect of chain length on catalytic efficiency. The largest eflFects were observed with substrates having three to four... 

This ambiguity was resolved in the experiment shown in Table XL The peptide in this case represents the C-terminal sequence of the B-chain of insulin. The major products were the penta-peptide lacking the N-terminal phenylalanine and free phenylalanine indicating that hydrolysis was the major reaction. In addition, however, there was a significant amount of Phe-Phe. This dipeptide could only come from a transpeptidation involving an acyl transfer. The acyl transfer presumably proceeds via a covalent acyl intermediate. The evidence for this comes from the experiment shown in Table XII where Leu-Tyr-Leu was incubated with both porcine pepsin and penicillopepsin in the presence of a lO-fold excess of C-leucine over Leu-Tyr-Leu. The products, leucine and leucylleucine, were separated by high voltage electrophoresis and analyzed for their specific radioactivity. At most, only traces of radio-... 

Insulin peptides. X. Synthesis of the B-chain of insulin and its combination with natural or synthetic A-chain to generate insulin activity. J. Am. Chem. Soc. 1964 86 930-932. 113. 

In a given protein inaccessibility of tryptophan units to NBS may not necessarily mean stability to enzymatic cleavage. In TMV-protein, for instance, all three tryptophan peptide bonds are cleaved by the action of chymotrypsin. This means that the approach of a chemical cleaving agent and of an enzjnne is affected by the same enviromental factors to a different degree. Exclusive iodination of the A chain of insulin is a related example of an unreactive tyrosine residue in the B chain (Springell, 1961). 

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