| Abstract: | Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite. |
