Enzymatic Synthesis of Hydroxyproline by the Hydroxylation of Poly(L-prolyl-glycyl-L-prolyl)

Abstract

[C14]Proline labeled polytripeptides of the structure [C14]Pro-Gly-Pro)n were prepared and fractionated. Fractionation of the polytripeptide preparations on a Sephadex G-50 column gave fractions ranging in molecular weight from about 1,700 to about 17,000. The helical content of various polytripeptide fractions was estimated by measure-ments of both optical rotation and optical rotatory dispersion (from the magnitude of the trough at 213 m[mu]). The degree of helicity in-creased markedly with the size of the polytripeptide fractions over the range of 1,700 to 8,000 or 10,000 molecular weight units. The relative increase in degree of helicity was less when fractions of 10,000 and 16,000 molecular weight were compared. The results were consistent with earlier reports which suggested that the poly-peptide (Pro-Gly-Pro)n in solution formed a triple helix of 1, 2, or 3 polypeptide strands similar to the 3-stranded triple helix of collagen, and that the amount of triple helix was greater in prepara-tions with larger molecular size. Fractions of [C14]Pro-Gly-Pro) were incubated with chick embryo extract, and a net synthesis of [C14]hydroxyproline was observed. In some experiments up to 16% of the [C14]proline in the labeled polytripeptide was converted to [C14]hydroxyproline. Although some variation in the activity of the hydroxylating system was observed from experiment to experiment, larger polymer fractions consistently gave higher yields of [C14] hydroxyproline than smaller fractions. The results suggest that the enzymatic system for the synthesis of collagen hydroxyproline re-quires proline-rich polypeptides which resemble collagen in amino acid sequence and which are of considerable size. The requirement for polypeptides of considerable size appears to constitute an un-usual feature of an enzymatic reaction and it is difficult to offer any explanation for this requirement. One possible explanation, however, is suggested by the parallel observed between the relative amount of helix and the amounts of [Cl4]hydroxyproline synthesized with (Pro-Gly-Pro)n fractions of different size. It will be of interest to examine further the hypothesis that the hydroxylase for the syn-thesis of collagen hydroxyproline requires a polypeptide substrate with a helical structure similar to collagen.