Biochemistry of Amino Acids - A. Majster 1961

Intermediary Metabolism of Amino Acids
Oxyproline

L-Hydroxyproline has been found in the acid hydrolyzates of only a few Proteins, notably Collagen and, in smaller amounts, Elastin. Its occurrence in nature appears to be largely restricted to animal Connective Tissue proteins. The Role of hydroxyproline in The Structure of collagen has been reviewed by Gustafson [1094]. To date, no organisms have been identified that require this amino acid for growth. When Sarcina lutea is grown in the presence of hydroxyproline, The amino acid accumulates within the Cells and in the culture medium of this microorganism [374]. allo-L-Hydroxyproline has been found in free form in sandalwood and in the acid hydrolyzates of phalloidin (p. 61). Recently, hydroxyproline has been detected in the acid hydrolyzates of proteins from carrot tissue grown in tissue culture [1095].

Following the administration of Proline labeled with N¹⁵ and deuterium to rats for three days, hydroxyproline was found to be the most heavily labeled amino acid among those isolated from the carcass (excluding proline itself). The N¹⁵:D ratio in the isolated hydroxyproline indicated that The conversion of proline to hydroxyproline was accompanied by the loss of approximately half of the deuterium bound to the carbon atoms of the proline molecule [357]. These findings point to a direct conversion of proline to hydroxyproline, though they do not rule out the existence of alternative biosynthetic pathways for hydroxyproline. When racemic N¹⁵-hydroxyproline was fed to rats, only about 0.3% of the hydroxyproline isolated from the carcass contained the label [375]. These results are consistent with the slow turnover rate of collagen (p. 274); however, administration of labeled proline resulted in a significantly higher isotope incorporation into carcass hydroxyproline than did feeding labeled hydroxyproline. Dietary labeled nitrogen from hydroxyproline was transferred to many body Amino Acids, most notably glutamic and aspartic acids. These data indicate a rapid breakdown of hydroxyproline in the rat Organism. Based on these findings, Stetten [375] suggested that the bulk of hydroxyproline in animal Tissues is likely formed from proline incorporated into peptide chains rather than from free hydroxyproline.

Witkop and coworkers [1096, 1097] investigated various compounds that might be involved in proline and hydroxyproline METABOLISM, including y-hydroxyornithine and y-ketoproline:

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Witkop and Beiler [1097] chemically achieved the conversion of y-hydroxyornithine stereoisomers into the corresponding hydroxyproline isomers.

It is highly probable that hydroxyproline is formed from proline in the rat; the reverse reaction apparently does not take place to any significant extent, since hydroxyproline, unlike proline, fails to replace Arginine in a diet designed to support optimal growth in young rats [376]. Hydroxyproline does not support the growth of proline-requiring mutants of Escherichia coli [377]. Meanwhile, it has been found that in this organism, hydroxyproline competes with glucose in proline synthesis [239]. The Nature of this interesting effect remains unclear. If the hydroxyproline preparations used in these experiments contained a small amount of proline impurity, this alone could account for the observed results.

It has long been established that hydroxyproline exhibits glycogenic activity [379, 380]. In vitro data point to its conversion into Glutamic Acid and Glutamine [359]. Stetten's studies [357–375] are consistent with this view. Rats were administered DL-hydroxyproline labeled with C¹⁴ at the a-carbon atom. Among the amino acids isolated from the tissues, the highest specific activity was found in glutamic acid; aspartic acid and hydroxyproline contained roughly equal amounts of the isotope, whereas proline contained very little. In glutamic acid, the highest isotope concentration was localized in the a-carbon atom; these data indicate the conversion of hydroxyproline to glutamic acid [381]. Recent studies with labeled hydroxyproline have suggested the existence of two metabolic pathways for this amino acid: one leading to The formation of glutamic acid, and the other to the formation of Alanine or a closely related compound [1098].

Adams [1091] studied hydroxyproline metabolism in an adapted strain of Pseudomonas. In soluble extracts from this organism, both L-hydroxyproline and allo-D-hydroxyproline were converted to glutamic acid. D-Hydroxyproline and allo-L-hydroxyproline underwent virtually no transformation. Only half of the utilized hydroxyproline was recovered as glutamic acid.

Studies using Liver and Kidney preparations have shown that L-proline is oxidized via glutamic acid to CO₂ and NH₃. The enzyme catalyzing this process, proline oxidase, also acts on L-hydroxyproline [367, 1092] and appears to be distinct from L-Amino Acid Oxidase. Hydroxyproline is not completely oxidized in this system; instead, an intermediate accumulates whose 2,4-dinitrophenylhydrazone is believed to be a derivative of y-hydroxyglutamic acid y-semialdehyde. Available data do not yet clarify the Nature of the intermediates formed during the conversion of hydroxyproline to glutamic acid.

Kidney D-Amino Acid Oxidase oxidizes D-proline to a-keto-d-aminovaleric acid [365] and, in the absence of catalase, to y-aminobutyric acid [382]; by contrast, The oxidation of D-hydroxyproline or allo-D-hydroxyproline in the presence of catalase yields pyrrole-2-carboxylic acid [382]. The formation of pyrrole-2-carboxylic acid from the oxidation intermediate (presumably the corresponding a-keto acid in equilibrium with its cyclized form) is a non-enzymatic, acid-catalyzed process:

Pyrrole-2-carboxylic acid has also been found among the breakdown products of mucoproteins and sialic acid [383, 1099].

Fig. 14. Summary scheme of hydroxyproline transformations.