Conformational aspects of proline hydroxylation in collagen biosynthesis : studies with synthetic peptides

Atreya, Prabhakara Lakshmi (1987) Conformational aspects of proline hydroxylation in collagen biosynthesis : studies with synthetic peptides. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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    Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
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Abstract

The hydroxylation of selected proline residues by prolylhydroxylase (E.C. 1.14.11.2) is a crucial posttranslational event in the biosynthesis of collagen, an important protein of the connective tissues. Hydroxyproline (Hyp) offers additional stability to the unique triple-helical conformation of collagen, which in turn is necessary for the functional viability of the protein, at physiological temperatures. -- Earlier studies on the substrate specificity of prolylhydroxylase have been intriguing. It was proposed earlier that prolylhydroxylase recognized the folded β-turn conformation, formed at the Pro-Gly segments in the nascent procollagen chains (Brahmachari and Ananthanarayanan, 1979). The present thesis involves the further elucidation of conformational aspects of proline hydroxylation in vitro, using chicken prolylhydroxylase and Pro-containing synthetic peptides. -- Pure prolylhydroxylase was obtained from 13-day old chicken embryos using established procedures. Pro-containing linear oligopeptides were characterized in different solvents, using circular dichroism (CD) and infrared (IR) spectroscopy. These studies have indicated the existence of two conformation, namely an extended conformation similar to that of poly (Pro) (PP-II) and a folded β-turn, in these peptides. The interaction between the enzyme and the oligopeptides of known conformation was studied by the following reactions: (1) hydroxylation of the peptides themselves and (2) the capability of these peptides to compete with the standard substrate, for the active site of prolylhydroxylase. It was found that peptides with either β-turn or extended conformation alone can act only as inhibitors. On the other hand, peptides with both these conformations can also serve as substrates for the enzyme, in addition to being competitive inhibitors. -- Based on these observations, a model is proposed for the conformational criteria of enzymatic proline hydroxylation. According to this model, the enzyme requires the presence of PP-II like extended conformation followed by folded β-turns in the substrate molecules. The PP-II structure is necessary at the binding site of the enzyme, while the β-turn structure is necessary at the catalytic site. Peptides with either one of these structures can act only as inhibitors since they can fulfill only part of the conformational requirement. These studies are of importance since they help to define the observed substrate specificity of prolylhydroxylase, in precise conformational terms. -- The structure-function relationship of the prolylhydroxylase itself and it interaction with substrates and cosubstrates, in conformational terms, are also studied by CD and fluorescence spectroscopy. The implications of these studies in understanding the substrate specificity of prolylhydroxylase are discussed.

Item Type: Thesis (Doctoral (PhD))
URI: http://research.library.mun.ca/id/eprint/4041
Item ID: 4041
Additional Information: Bibliography: leaves 291-305.
Department(s): Science, Faculty of > Biochemistry
Date: 1987
Date Type: Submission
Library of Congress Subject Heading: Proline; Hydroxylation; Proline hydroxylase

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