Y-Hydroxylysine

Concentrated hydrochloric acid cleaved the peptide chain after the y-hydroxylysine residue. Edman degradation of these various peptides permitted determination of the complete sequence of amino acids and led to proposal of structure (35) for cerexin A 85). Further work (55) showed that the cerexin fraction could be resolved into ten components, the structures of which were established (Table 4). 

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... 

Elastin confers extensibihty and elastic recoil on tissues. Elastin lacks hydroxylysine, Gly-X-Y sequences, triple hehcal stmcture, and sugars but contains desmosine and isodesmosine cross-links not found in collagen. 

Although the exact amino acid sequence differs between the various collagens, the primary structure usually conforms to a repeating tripeptide Gly-X-Y where X and Y are, proline, lysine, or hydroxyproline, hydroxylysine respectively. A single unit of collagen is a triple helix composed of three a chains. This conformation differs from the common a helix found in proteins in two important ways ... 

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. 

Cofactor for hydroxylation reactions, for example In procollagen Proline -> hydroxyproline Lysine -> hydroxylysine —Ot.QrT Y i —. ft... 

Collagen is rich in glycine and proline. The glycine residues are part of a repeating sequence, -Gly-X-Y-, where X is frequently proline and Y is often hydroxyproline or hydroxylysine. 

Leucine oxidized with "OH produces a 5 -hydroperoxy derivative which is subjected to chemical reduction to yield (2S)-y-hydroxyleucine, (2S,4S)-8-hydroxy-leucine, (2S,4R)-8-hydroxyleucine. The S -hydroxyleucines have been confirmed to be the reduction products of the corresponding hydroperoxyleucines. 5 -Hydro-xylysines are natural products formed by lysyl oxidase and therefore are not useful markers of radical-mediated oxidation. The other hydroxylysines are useful markers, however, with HPLC analysis of 9-fluorenylmethyl chloroformate (FMOC)... 

Due to an increased interest in analysis of physiological samples, we wanted to establish analyzer methods which would allow us to choose between our standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids, to include the most important free amino acids found in physiological samples. A study of common analysis requirements in our facility indicated that only a limited number of the possible free physiological amino acids is needed for most unknown samples. These additional amino acids of interest are a-amino butyric acid, citrulline, y-amino butyric acid (GABA), hydroxyproline, hydroxylysine, ornithine, taurine, and tryptophan. Other amino acids of interest to us are phosphoserine, phosphothreonine, phosphotyrosine and carboxy-amino acids since they are released from glycoprotein or glycopeptide hydrolysates. 

Y. Kakimoto and S. Akazawa, Isolation and Identification ofNG, NG-Dimethylarginine, N-Epsilon-Mono-, Di-, and Trimethyllysine, and Glucosylgalactosyl- and Galactosyl-Delta-Hydroxylysine From Human Urine, Journal of Biological Chemistry 245 (1970) 5751 -5758. 

Side-chain CH2 may be hydroxylated, e.g. hydroxylysine, hydroxyprolines (trans-4-hydroxyproline in particular), or carboxylated, e.g. to give ot-aminomalonic acid, (3-carboxyaspartic acid, y-carboxyglutamic acid, (3-hydroxyaspartic acid, etc. 

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 tropocollagen [5- and y-chains in Fig. 4.1 are respectively twice and three-times the size of the a-chain. Indeed, their amino acid composition is virtually identical to the a-chain in Fig. 4.1, except for the appearance of derivatives of lysine and hydroxylysine, mainly lysyl hydroxyl-norleucine and pyridinoline. They increase in amount with age of the organism. 

Hydroxylysine glycosides occur at the Y-position and may play a role in determining fibril diameter. The side chains of these amino acids project outward from the center of the triple helix, permitting hydrophobic and ionic interactions between tropocollagen molecules. These interactions determine the manner in which individual tropocollagen molecules aggregate to form microfibrils initially, then larger fibrils, and eventually fibers. 

Mature elastin is a linear polypeptide, tropoelastin, which has a molecular weight of about 72,000 and contains about 850 amino acid residues. Although glycine accounts for one third of the residues, the repeat sequence Gly-X-Y characteristic of collagen is not present in elastin. Instead, glycine residues are present in the repeat units Gly-Gly-Val-Pro, Pro-Gly-Val-Gly-Val, and Pro-Gly-Val-Gly-Val-Ala. Elastin is relatively rich in the nonpolar amino acids alanine, valine, and proline. In contrast to collagen, only a few hydroxyproline residues are present in elastin. Elastin contains no hydroxylysine or sugar residues. 

Collagen s amino acid sequence primarily consists of large numbers of repeating triplets with the sequence of Gly—X—Y, in which X and Y are often proline and hydroxyproline. Hydroxylysine is also found in the Y position. Simple carbohydrate groups are often attached to the hydroxyl group of hydroxylysine... 

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