Studies on protocollagen lysine hydroxylase. Hydroxylation of synthetic peptides and the stoichiometric decarboxylation of -ketoglutarate. Academic Article uri icon


  • A series of synthetic peptides was prepared and used to study protocollagen lysine hydroxylase, the enzyme which synthesizes the hydroxylysine in collagen. The enzyme was purified 150- to 250-fold from chick embryo extracts and it was completely free of protocollagen proline hydroxylase activity. Several synthetic peptides competitively prevented the hydroxylation of lysine-labeled [14C]protocollagen by protocollagen lysine hydroxylase. When the peptides were examined directly as substrates for the enzyme, the synthesis of measurable amounts of hydroxylysine was observed for the first time. The synthesis of hydroxylysine was accompanied by the conversion of a-ketoglutarate to C02 and succinate, indicating that the enzyme belongs to the new class of oxygenases which decar-boxylate a-ketoglutarate during substrate hydroxylation. The release of [14C]CO-2 from [l-14Ci]a-ketoglutarate was equimolar with the synthesis of hydroxylysine and therefore the amount of [14C]C02 released could be used as a simple assay for the enzymic reaction. Three peptides were synthesized which had amino acid sequences comparable to amino acid sequences around glycosylated hydroxylysine in collagen. The peptide L-I had the sequence Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly, L-II the sequence Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4, and L-IH the sequence (Pro-Pro-Gly)i-Ala-Arg-Gly-Met-Lys-Gly-His-Arg - Gly - (Pm - Pro- Gly)4. When the peptides were used as substrates for protocollagen lysine hydroxylase, the Fmax for all three peptides was the same. The Km for L-I was twice the Km for L-II but there was no difference in the Km for L-II and L-III. In contrast, when L-II and L-III were compared as substrates for protocollagen proline hydroxylase, the Km for L-II was three times the K, l for L-III. Free lysine and the tripeptide Lys-Gly-Pro did not serve as substrates for the synthesis of hydroxylysine. A very slow rate of hydroxylysine synthesis was observed with the tripeptide Ile-Lys-Gly, whereas the rate observed with the hexapeptide (Ile-Lys-Gly)2 was over ten times greater. Also, lysine8-vasopressin with the terminal sequence -Cys-Pro-Lys-Gly-NH2 was a substrate for the synthesis of hydroxylysine. However, the Vnmx and Km values for (Ile-Lys-Gly)2 and lysine8-vasopressin indicated that they were poorer substrates than L-I, L-II, or L-III. Reduced and carboxymethylated collagen from the cuticle of Ascaris lumbricoides and peptides derived from this collagen did not interact with the enzyme, even though the collagen contains about 40 residues of lysine/1000 residues and no hydroxylysine. The data obtained with the various peptides indicated that both the amino acid sequence around lysine and the length of the chain are critical determinants in the synthesis of hydroxylysine by protocollagen lysine hydroxylase. 1972, American Chemical Society. All rights reserved.

published proceedings

  • Biochemistry

altmetric score

  • 3

author list (cited authors)

  • Kivirikko, K. I., Shudo, K., Sakakibara, S., & Prockop, D. J.

citation count

  • 78

complete list of authors

  • Kivirikko, KI||Shudo, K||Sakakibara, S||Prockop, DJ

publication date

  • January 1972