title: Correlating crosslink formation with enzymatic activity in cysteine dioxygenase.
abstract: Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40-50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.
authors.affiliation: Department of Chemistry, University of Otago, Dunedin, New Zealand.
authors.forename: Eleni
authors.initials: E
authors.lastname: Siakkou
authors.forename: Malcolm T
authors.initials: MT
authors.lastname: Rutledge
authors.forename: Sigurd M
authors.initials: SM
authors.lastname: Wilbanks
authors.forename: Guy N L
authors.initials: GN
authors.lastname: Jameson
mesh_terms: D000332:Aerobiosis / Q000502:physiology; D000818:Animals; D005591:Chemical Fractionation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D050558:Cysteine Dioxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D007501:Iron; D050558:Cysteine Dioxygenase