Marrone, Laura2006-07-282006-07-2819971997http://hdl.handle.net/10012/96Recombinant lysine:N6 -hydroxy lase catalyses the conversion of L-lysine to its N6- hydroxy derivative upon supplementation with cofactors NADPH and FAD. The catalytic function of the protein is adversely affected at higher concentrations of c1· ions, with total loss of activity being observed at concentrations ~600 mM of these ions. In contrast~ under similar ionic strength conditions, both phosphate and sulfate ions have no such deleterious effects on the enzyme. Of the five cysteine residues present in the protein, three are accessible to titration with 5,5'-dithiobis (2-nitrobenzoic acid), DTNB, in the native conformation of the protein. In contrast, under similar experimental conditions only two functions are alkylatable by iodoacetate and these have been identified as CysS l and Cys 158 residues present in the protein. Modification of thiol groups either by DTNB or iodoacetate results in a complete loss of the protein's catalytic function. rlucD can form either a covalent or noncovalent complex with 2,6-dichlorophenol indophenol (DPIP), the former process being dependent on the presence of unmodified thiol functions in the protein. Both the covalent and the noncovalent complexes of rlucD and DPIP are capable of mediating NADPH oxidation by a mechanism involving an exchange of reducing equivalents between the protein bound dye and that free in the environment. However, only the latter type of complex which is formed in the absence of thiol groups in rlucD is capable of functioning as a diaphorase in the presence of FAD. The replacement of CysS l and Cys 158 of riucD with alanine residues, by site directed mutagenesis ofiucD, does not lead to a loss in the catalytic function(s) of the protein. Studies with rlucD muteins have shown that CysS l plays an important role in the protein's covalent interaction with DPIP. Replacement of CysSI and Cysl58, either individually or in combination, with alanine is accompanied by an enhancement in the ability of rlucD to promote NADPH oxidation in the absence of its hydroxylatable substrate. rlucD is stringently specific with respect to its hydroxylatable substrate. This feature has formed the basis for a novel proposal which envisages the participation of an activated I act am intermediate of the substrate in the catalytic mechanism of the protein.application/pdf8351461 bytesapplication/pdfenCopyright: 1997, Marrone, Laura. All rights reserved.Harvested from Collections CanadaDoctoral Thesis