Prolyl and lysyl residues

The active form of vitamin C is ascorbate acid (Figure 28.8). The main function of ascorbate is as a reducing agent in several different reac lions. Vitamin C has a well-documented role as a coenzyme in hydroxy lation reactions, for example, hydroxylation of prolyl- and lysyl-residues of collagen (see p. 47). Vitamin C is, therefore, required for the mainte nance of normal connective tissue, as well as for wound healing. Vitamin C also facilitates the absorption of dietary jron from the intestine. 

Ascorbic acid has been shown to increase collagen synthesis by fibroblasts in vitro (99, 280) and to maintain collagen synthesis In nonmitotic fibroblEists for extended periods (99). Prolyl hydroxylase, the enzyme hydroxylating prolyl and lysyl residues of procollagen, requires ascorbate to function in vitro (181), and the addition of ascorbic acid to tissue cultures stimulates the prolyl hydroxylase activity of fibroblasts (174). 

The intracellular protein-making machinery first of all produces polypeptide a-chains (approximately 1000 amino acids). Some of the prolyl and lysyl residues are hydroxylated by reactions that need vitamin C (Chapter 56). Some of the hydroxylysyl residues are glycosylated. The units then associate to form the triple helix (rope-like) procollagen, which is soluble. 

One of the most important functions so far attributed to ascorbate is the acceleration of hydroxylation reactions in a number of biosynthetic pathways (recently reviewed by Englard and Seifter, 1986 Padh, 1990). Among these pathways, the hydroxylation of prolyl and lysyl residues in the biosynthesis of collagen is most... 

Hydroxylation of prolyl and lysyl residues occurs mainly in collagen, elastin, and some plasma proteins. Hydroxylysines and hydroxyprolines have been found also in acetylcholinesterase and in the C q subcomponent of complement (see review, Kivirikko and Myllyla, 1980). In collagen, the role of hydroxylation seems to consist in stabilization of the triple helix. 

Fig. 7.4 Catalytic action of proline hydroxylase. One atom of oxygen gas (red) oxidizes ketoglutarate and appears in succinate along with COr The other oxygen atom forms a Fe3+-OH complex attached to the enzyme. When Fe3+ is reduced by ascorbate, H+ dissociates and the O-1 species (red) is released to a proline residue in the polypeptide, forming a hydroxyproline residue. The enzymes recognize prolyl- or lysyl residue sequence motifs described in the text (From Fig. 11.3 in Biochemistry, L. Stryer,...

Hydroxylation of prolyl residues and some lysyl residues glycosylation of some hydroxylysyl residues... 

This enzyme [EC 3.4.14.1], also called cathepsin C and cathepsin J, catalyzes the hydrolysis of a peptide bond resulting in the release of an N-terminal dipeptide, XaaXbb-Xcc, except when Xaa is an arginyl or a lysyl residue, or Xbb or Xcc is a prolyl residue. This enzyme, a member of the peptidase family Cl, is a CF-dependent lysosomal cysteine-type peptidase. 

Systematic modification of the octapeptide sequence from cin-giotensinogen (Figure 1) was undertaken to incorporate desirable properties into the peptide. Addition of a prolyl residue to the N-termlnus Improved solubility at physiologic pH, replacement of the leucyl-leucine sequence with phenylalanyl residues Improved inhibitory properties by forty-fold, and addition of a lysyl residue to the C-termlnus Increased solubility and extended half-life in vivo. These modifications yielded the Renin Inhibitory Peptide TriP) vdiich effectively blocks renin both in primates (11) and man (1 2). 

AA serves as an important cofactor for enzymes. Lack of AA in food causes scurvy in humans due to inefficient collagen synthesis, caused by the inactivation of the Fe(II)-activating prolyl hydroxylase and lysyl hydroxylase which catalyze the formation of hydroxyproline and hydroxylysine as essential components for collagens. Prolyl hydroxylases can also hydroxylate conserved prolyl residues in the alpha subunit of the hypoxia-inducible transcription factor, which signals for proteasomal degradation of the transcription factor . The proper action of these hydrolases requires dioxygen, thus they can act... 

The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxy-peptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group at the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963). 

Conversions of peptide-prolyl and peptide-lysyl residues to hydroxyprolyl and hydroxylsyl residues are accomplished by iron-dependent enzymes utilizing... 

Procollagen(I) is an example of a protein that undergoes extensive posttransla-tional modifications. Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues. These reactions occur after the protein has been synthesized (Fig. 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes, for example, prolyl hydroxylases and lysyl hydroxylase. Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose). 

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