Of 5-hydroxylysine

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

Before this "maturation" can occur there must be other modifications to procollagen. These begin while the peptide chains are still attached to ribosomes of the rough ER. Hydroxylases (Chapter 18) localized in the membranous vesicles of the ER convert some of the proline and lysine residues of the procollagen chains into 4-hydroxyproIine625 629 630 and hydroxylysine (Eqs. 8-6 and 8-7). Lesser amounts of 3-hydroxypro-line are formed. About 100 molecules of 4-hydrox-yproline and 50 of 5-hydroxylysine are created in each al chain. 

The glycosyltransferase which glycosylates the hydroxyl group of 5-hydroxylysine in collagens and similar proteins appears to recognize the amino acid sequence -G1 y-X—HyZ-Gly-Y-Arg- Whereas the amino acids X and Y are quite variable, the positions of Gly, Hyl, and Arg are invariant in the glycopeptides of ten vertebrate and invertebrate collagens (169). 

Hydroxylated amino acids (eg, 4-hydroxyproline, 5-hydroxylysine) and A/-methylated amino acids (eg, /V-methylhistidine) are obtained by the acid hydrolysis of proteins. y-Carboxyglutamic acid occurs as a component of some sections of protein molecules it decarboxylates spontaneously to L-glutamate at low pH. These examples are formed upon the nontranslational modification of protein and are often called secondary protein amino acids... 

Benzyl-oxygen bonds may be cleaved under conditions mild enough to leave an allylic hydroxy group (759) or an easily reduced N—OH bond intact (65,80). N-Hydroxyamino acids can be prepared in good yield by hydrogenolysis of benzyl hydroxamates as shown in the synthesis of N -hydroxylysine (6) from 5 (777). 

Vitamin C is essential for the formation of collagen, the principal structural protein in skin, bone, tendons, and ligaments, being a cofactor in the hydroxylation of the amino acids proline to 4-hydroxyproline, and of lysine to 5-hydroxylysine. These hydroxyamino acids account for up to 25% of the collagen structure. Vitamin C is also associated with some other hydroxylation reactions, e.g. the hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) in the pathway to catecholamines (see Box 15.3). Deficiency leads to scurvy, a condition characterized by muscular pain, skin lesions, fragile blood vessels, bleeding gums, and tooth loss. Vitamin C also has valuable antioxidant properties (see Box 9.2), and these are exploited commercially in the food industries. 

The triplet Cly-X-Y is constantly repeated in the sequence of the triple-helical regions— i. e., every third amino acid in such sequences is aglycine. Proline (Pro) is frequently found in positions X or Y the Y position is often occupied by 4-hydroxyproline (4Hyp), although 3-hydroxyproline (3Hyp) and 5-hydroxylysine (5Hyl) also occur. These hydroxylated amino acids are characteristic components of collagen. They are only produced after protein biosynthesis by hydroxylation of the amino acids in the peptide chain (see p. 62). 

In addition to the 20 common amino acids, proteins may contain residues created by modification of common residues already incorporated into a polypeptide (Fig. 3-8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-N-Methyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is y-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their biological function. More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. 

Fig. 5. Synthesis of 1,2-trans glycosides using a participating group at C-2 for example, synthesis of glycosylated hydroxylysine derivative (p-D-Gal-( 1 >0)-IIyl) using (a) insoluble promoter-silver silicate (56) (b) soluble promoter-silver trifluo-romethanesulfonate (57).

One-third of the amino acid residues in collagen are Gly, while another quarter are Pro. The hydroxylated amino acids 4-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl) are formed post-translationally by the action of proline hydroxylase and lysine hydroxylase. These Fe2+-containing enzymes require ascorbic acid (vitamin C) for activity. In the vitamin C deficiency disease scurvy, collagen does not form correctly due to the inability to hydroxylate Pro and Lys. Hyl residues are often post-translationally modified with carbohydrate. 

In addition to the standard amino acids, other amino acids are found in protein in smaller quantities. For example, 4-hydroxyproline and 5-hydroxylysine are hydroxylated versions of standard amino acids. These are called rare amino acids, even though they are commonly found in collagen. 

After the secretion into the extracellular space, collagen molecules may be modified chemically even more. Some collagen processing enzymes cleave the procollagen molecules to mature tissue type molecules. Lysine and 5-hydroxylysine residues can be modified enzymatically to allysine (alpha-aminoadipic-acid delta-semialdehyde) and hydroxyallysine (delta-hydroxy, alpha-aminoadipic acid delta-semialdehyde), respectively, by lysyl oxidase (protein-lysine 6-oxidase) (EC 1.4.3.13). The aldehyde group then interacts with the amino group of an adjacent lysine residue to form a Schiff base. Many... 

Several other types of covalent crosslinks, mostly derived from lysine or 5-hydroxylysine residues (the latter being formed by post-translational modification), are found in collagen and elastin. A few examples are given (5.2-5.7) A6 7-dehy-drolysinonorleucine (5.2), lysinonorleucine (5.3), dehydrohydroxylysinonorleucine (5.4), lysino-5-ketonorleucine (5.5), desmosine (5.6) and isodesmosine (5.7). An intrachain thiol ester loop is present in a2-macroglobulin and proteins of the complement system and consists of a fifteen-membered ring derived from cysteine and glutamic acid (5.8). 

Another example of a peptide sequence in a protein forcing it into a biologically useful conformation is found with collagen. This consists of a triple helix with chains of more than 1000 amino-acid residues, many of which are post-translation-ally modified. The latter steps, consisting inter alia of hydroxylation of Pro and Lys residues and 5-hydroxylysine residues, occur before the triple helix is formed, because the enzymes involved do not act on the helical structure. When the individual peptide chains of collagen are synthesised, there are N- and C-terminal sequences each containing about 100 amino-acid residues. These sequences favour the formation of a triple helix. When this has been achieved, the terminal sequences are removed. The sequences of these temporary terminal sequences are quite different from the main body of the collagen monomers, which consists of triads of the type Gly—X—Y, where X and Y are often proline or 3- or 4-hydroxyproline. 

The amino acid composition of collagen is distinctive. Glycine constitutes approximately one-third of the amino acid residues. Proline and 4-hydroxyproline may account for as much as 30% of a collagen molecule s amino acid composition. Small amounts of 3-hydroxyproline and 5-hydroxylysine also occur. (Specific proline and lysine residues in collagen s primary sequence are hydroxylated within the rough ER after the polypeptides have been synthesized. These reactions, which are discussed in Chapter 19, require the antioxidant ascorbic acid.)... 

Two unusual, hydroxylated amino acids account for nearly one-fourth of the amino acids in collagen. These amino acids are 4-hydroxyproline and 5-hydroxylysine. 

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