| The biosynthesis of collagen involves several post-translational modifying steps which are critical in the ultimate formation, diversification and conferment of structural integrity to the mature collagen fiber. Lysyl hydroxylase is a collagen post-translational modifying enzyme located in the lumen of the endoplasmic reticulum which hydroxylates lysine residues at X-Y-Gly motifs on the nascent collagen polypeptide. Organophosphate pesticides such as malathion and its oxidized metabolite malaoxon are better known for their neurotoxic effects on acetylcholinesterase, although the foundation of this work indicates that these pesticides have lathrogenic effects as well. In this dissertation, malathion and malaoxon are shown to be direct inhibitors of lysyl hydroxylase. The effects of these compounds were initially explored in cell cultured rat fetal lung fibroblasts exposed to doses ranging from 0–125μM of malathion and malaoxon. In the absence of any measurable effects on cytotoxicity, total collagen production, collagen type I/III shifting or the level of lysyl hydroxylase protein expression, significant decreases in the hydroxylysine content of acid hydrolyzed cell lysates from these exposed cultures were found. In addition, a direct inhibition of lysyl hydroxylase by malathion and malaoxon was found using an in vitro enzyme assay, in which the IC50's for malathion and malaoxon were calculated to be 60μM and 45μM respectively. Further inhibition kinetic studies were carried out, in which malathion was found to be a partial competitive inhibitor with respect to a procollagen-like synthetic peptide substrate and malaoxon was found to be a pure competitive inhibitor with respect to the same substrate. Both compounds were found to be partial uncompetitive inhibitors with respect to the Fe+2 cofactor and partial noncompetitive inhibitors with respect to ascorbic acid.; Site-directed mutagenesis of seven residues (H657, H709, D659, H707, H614, H658 and D675) in the C-terminal region of rat lysyl hydroxylase believed to be important in catalytic activity was employed to gain a better understanding of residues important in structure and function of this enzyme. All the residues mutated were found to inactivate the enzyme with the exception of H614, which was the only residue not conserved in the lysyl hydroxylase sequence of other species. |
