LYSINE-N-6-HYDROXYLASE - COFACTOR INTERACTIONS

Recombinant lysine: N-6-hydroxylase (rIucD) requires the cofactors FAD and NADPH for its catalytic function of the conversion of L-lysine to its N-6-hydroxy derivative. In the presence of high concentration of chloride ions (greater than or equal to 600 mM), the protein exists in a reversible inactive conformation. Depending on the oxidation state of its thiol functions, rIucD can bind 2,6-dichlorophenol indophenol ( DPIP), either covalently or noncovalently, the former type of interact ion occurring with protein preparations possessing unmodified thiol gr oups. Both covalent and noncovalent complexes of rIucD and DPIP appear to be capable of NADPH oxidation in the presence of exogenous DPIP by a phenomenon of exchange of reducing equivalents between the protein- bound dye and that free in the medium. In the presence of FAD, the lat ter type of complex has been found to function as a diaphorase. The di minution in the catalytic activity of rIucD observed at high concentra tions of the flavin cofactor does not appear to be due to an uncouplin g of the processes of NADPH oxidation and lysine: N-hydroxylation caus ed by an exchange of reducing equivalents between the enzyme-bound FAD and that free in the medium. (C) 1996 Academic Press, Inc.