Zijenah, Lynn Sodai (1986) Syntheses and conformation of synthetic peptide substrates of protocollagen lysyl hydroxylase. Masters thesis, Memorial University of Newfoundland.
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Hydroxylation of specific lysyl residues by lysyl hydroxylase is an important posttranslational modification process in collagen biosynthesis. The main objective of this work was to investigate the conformational requirement for the enzymic reaction. -- Eight lysine-containing peptides which had amino acid sequences comparable to amino acid sequences around hydroxylysine or lysine in collagen were synthesized by solution-phase techniques. The peptides varied in length from three to seven amino acid residues. The structures of these peptides were investigated through circular dichroism (CD) and infrared (IR) spectroscopic methods. -- Lysyl hydroxylase was partially purified from chicken embryos using the established procedures. Seven of the synthetic peptides were tested for their ability to act as substrates of partially purified lysyl hydroxylase. The hydroxylation reaction was assayed by a technique involving measurement of ¹⁴CO₂ released stoichiometrically from 2-[1-¹⁴C]oxoglutarate and/or by a specific chemical procedure for hydroxylysine. -- Five peptides with the -Lys-Gly- sequence were hydroxylated to varying degrees, the degree of hydroxylation increasing with increasing chain length. Examination of these hydroxylated peptides by CD and IR spectral measurements revealed that the tripeptides NαtBocIleLysGlyOH and NαtBocAlaLysGlyOH adopt a γ-turn in which lysine occupies the second position of this structure. The tetrapeptide (NαtBocAlaLysGlySerOH) adopts both a β- and γ-turn and is more hydroxylated than the precursor tripeptide. This increase in the degree of hydroxylation may be attributed to the presence of the β-turn which may stabilize the γ-turn formed by the AlaLysGlyOH segment. The hexapeptide (NαtBocLeuHyPGlyAlaLysGlyOH) adopts a consecutive β- and γ-turn and is more hydroxylated than the tetrapeptide. This increase in hydroxylation may be attributable to the Gly³-Ala⁴ segment which may increase the binding of the enzyme to the substrate thereby enhancing hydroxylation. The heptapeptide (NαtBocLeuHyPGlyAlaLysGlySerOH) is hydroxylated more than the precursor hexapeptide. CD and model building studies have shown that NαtBocLeuHyPGlyAlaLysGlySerOH adopts two consecutive β-turns and a γ-turn. The second β-turn which is similar to that found in the tetrapeptide (NαtBocAlaLysGlySerOH) may be responsible for the increase in hydroxylation in comparison with the hexapeptide. -- All the hydroxylated peptides have one structural feature in common, namely the γ-turn with lysine in the second position. In contrast, two peptides (NαtBocAlaGlyLysOH and NαtBocAlaGlyLysHyPOH) which have the Gly-Lys sequence were not hydroxylated. Interestingly, both peptides adopt a γ-turn but the lysine is found in the third position of this structure. These data indicate that lysyl hydroxylase recognizes specific secondary structure(s) in its substrates. The nature of the amino acid around lysine and the chain length of the peptide may be the critical determinants in the synthesis of hydroxylysine by lysyl hydroxylase.
|Item Type:||Thesis (Masters)|
|Additional Information:||Bibliography: leaves 129-134.|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Hydroxylation; Collagen|
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