Proline residues hydroxylation

In humans as well as in other but not all mammalian species, kininogens are modified by posttranslational hydroxylation of a single proline residue of their kinin sequence, i.e. position 3 in bradykinin or position 4 in kallidin. Hydroxylation appears not to affect the specificity, affinity or intrinsic efficacy of the kinins. 

Since 1973, several authors have proved that there is a relationship between thermostability of collagen and the extent of hydroxylation of the proline residues31,34). Equilibrium measurements of the peptides al-CB 2 of rat tail and rat skin revealed a higher rm, for al-CB 2 (rat skin)157). The sequence of both peptides is identical except that in the peptide obtained from rat skin, the hydroxylation of the proline residues in position 3 has occurred to a higher extent than in the case of al-CB 2 (rat tail). Thus, a mere difference of 1.8 hydroxy residues per chain causes a ATm of 26 K. Obviously, there are different stabilizing interactions in the triple-helical state, that means al-CB 2 (rat skin) forms more exothermic bonds than al-CB 2 (rat tail) in the coil triple-helix transition. This leads to an additional gain of enthalpy which overcompensates the meanwhile occurring losses of entropy. 

Very few post-translational modifications have been found on tropoelastin. However, hydroxylation of 25% of the proline residues is observed [10]. The enzymatic modification of proline to hydroxyproline (Hyp) is performed by prolyl hydroxylase [11]. The purpose of this hydroxylation remains unclear and it is even proposed that Hyps in tropoelastin are a by-product of collagen hydroxylation as this occurs in the same cellular compartment [8]. 

Rather scanty evidence exists for the participation of free radicals in Alzheimer s disease and Down s syndrome. However, more recendy, reports have appeared that suggest possible free-radical involvement in the pathogenesis of these two conditions. Zemlan et al. (1989) repotted that the activity of the free-radical scavenging enzyme, SOD, was significantly increased in fibroblast cell lines derived from familial Alzheimer s and Down s patients. They hypothesized that the elevation in SOD activity observed in the Alzheimer patients supports the theory that paired helical filaments are formed by free-radical hydroxylation of proline residues. They further su ested that SOD levels might also be increased in the brains of Alzheimer s and Down s patients, and that the increase in SOD may reflect an enhanced generation of free radicals. 

Another important 2-OG dependent oxygenase in mammals is prolyl-4 hydroxylase, which catalyzes the hydroxylation of the proline residue in collagen (Scheme 5). This reaction is essential for the structure of the collagen triple helices (9,34 6). An overproduction of collagen is related to fibrotic diseases such as rheumatic arthritis. Thus collagen prolyl-4 hydroxylase is a target for therapeutics (34,36). 

Prolyl 4-hydroxylation is the most abundant posttranslational modification of collagens. 4-Hydroxylation of proline residues increases the stability of the triple helix and is a key element in the folding of the collagen triple helix. " In vertebrates, almost all the Yaa position prolines of the Gly-Xaa-Yaa repeat are modified to 4(I( )-hydroxylproline by the enzyme P4H (EC 1.14.11.2), a member of Fe(II)- and 2-oxoglutarate-dependent dioxygenases. This enzyme is an 0 2/ b2-type heterotetramer in which the / subunit is PDI (EC 5.3.4.1), which is a ubiquitous disulfide bond catalyst. The P4H a subunit needs the 13 subunit for solubility however, the 13 subunit, PDI, is soluble by itself and is present in excess in the ER. Three isoforms of the a subunit have been identified and shown to combine with PDI to form [a(I)]2/ 2) [< (II)]2/32> or [a(III)]2/32 tetramers, called the type... 

Ascorbic acid (vitamin C fig. 10.16) is the reducing agent required to maintain the activity of a number of enzymes, most notably proline hydroxylase, which forms 4-hydroxyproline residues in collagen. Hydroxyproline (see fig. 10.16c) is not synthesized biologically as a free amino acid but rather is created by modification of proline residues already incorporated into collagen. The hydroxylation reaction occurs as the protein is synthesized in the endoplasmic reticulum. At least a third of the numerous proline residues in collagen are modified in this way, substantially increasing the resistance of the protein to thermal denaturation. 

There are 20 different amino acids used in the synthesis of proteins these amino acids are listed in Table 3.1, which also contains the two commonly used symbols for each amino acid. The three-letter symbols are easier to remember, but the single-letter symbols are often used in writing long sequences. In many proteins some of the amino acids are modified after incorporation into proteins e.g., in collagen, a hydroxyl group is added to each of several proline residues to yield hydroxyproline residues. With the exception of proline, the a-amino acids that are incorporated into proteins can be represented by the formula shown in Fig. 3-1. 

The enzyme prolylhydroxylase catalyzes the hydroxylation of proline residues, which lie immediately before the repetitive glycine residues in the pro-a-chains ... 

The other major protein in the extracellular matrix is elastin, which is the main component of elastic fibers found in ligaments, large arteries, and lungs. After synthesis and partial hydroxylation of proline residues, a 72 kDa molecule of tropoelastin is secreted into the matrix. This protein is rich in nonpolar amino acids and contains repeating sequences, such as (Val-Pro-Gly-Val-Gly). These sections form an amorphous, random-coiled structure with frequent reverse turns. Other recurrent sequences are rich in alanine with paired lysine residues, e.g., -Ala-Ala-Ala-Ala-Lys-Ala-Ala-Lys-... 

Molecular mass 13,343 Da (nonhydroxylated) 13,359 Da (hydroxylated proline residue at position 3)... 

A20. Ayala, A., and Cutler, R. G., The utilization of 5-hydroxyl-2-amino valeric acid as a specific marker of oxidised arginine and proline residues in proteins. Free Radicals Biol. Med. 21,65-80 (1996). 

Analysis showed that the snail agglutinin contained a preponderance of acidic and hydroxylic amino acids and a large proportion of proline residues.83,560-562 Uncharacteristic of lectins from leguminous-plant seeds, the hemagglutinin contained 18 half-cystine residues and 10 molecular proportions of methionine per molecule of protein.83,569 About 8% (by weight) of covalently bound carbohydrate was found this was principally D-galactose and D-mannose.63... 

The first evidence for PDI having a different role came from Pihla-janiemi etal. (1987) during their study of the enzyme prolyl 4-hydroxylase (P4H). This enzyme is localized in the ER and catalyzes the hydroxylation of proline residues in nascent procollagen polypeptides. The enzyme is a tetramer composed of two a and two jS subunits (Kivirikko et al., 1989). Antibodies raised against the holoenzyme were used to screen a human gtll expression library and clones were isolated that coded for the /3 subunit. After sequencing it was revealed that the human /3 subunit was 94% identical with the rat PDI sequence. Southern blot analysis identified... 

After the peptide chain of collagen has been formed, many of the proline residues are hydroxylated on one of the ring carbon atoms. Why is this process important for the triple helix of collagen ... 

Vitamins are small biomolecules that are needed in small amounts in the diets of higher animals. The water-soluble vitamins are vitamin C (ascorbate, an antioxidant) and the vitamin B complex (components of coenzymes). Ascorbate is required for the hydroxylation of proline residues in collagen, a key protein of connective tissue. The fat-soluble vitamins are vitamin A (a precursor of retinal), D (a regulator of calcium and phosphorus metabolism), E (an antioxidant in membranes), and K (a participant in the carboxylation of glutamate). 

The most important reaction requiring ascorbate as a cofactor is the hydroxylation of proline residues in collagen. Vitamin C is, therefore, required for the maintenance of normal connective tissue as well as for wound healing since synthesis of connective tissue is the first event in wound tissue remodeling. 

Ascorbic acid is a cofactor in various hydroxylation reactions. These reactions include the hydroxylation of proline residues in a variety of proteins, such as the... 

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