FAD AND SUBSTRATE-ANALOGS AS PROBES FOR LYSINE N-6-HYDROXYLASE FROM ESCHERICHIA-COLI EN-222

Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal am ino function Of L-lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a K(d ) of 30 muM at 4-degrees-C. The spectral properties of FAD bound to ly sine N-hydroxylase are very similar to free oxidized FAD. In the absen ce of substrate, the enzyme has an NADPH oxidase activity which result s in the generation of hydrogen peroxide. With increasing concentratio n of L-lysine, the NADPH oxidase activity is enhanced up to 10-fold an d the generation of hydrogen peroxide decreases. At the same time, the substrate is hydroxylated. K(m) values for L-lysine and FAD were dete rmined as 105 muM and 0.7 muM, respectively. Utilizing FAD analogs, we could demonstrate that L-lysine exerts its effector role mostly on th e reductive half reaction of the overall catalytic cycle. Prolonged in cubation of the enzyme with either 8-chloro- or 8-fluoro-FAD gave rise to a covalently attached flavin which is formed as a result of the nu cleophilic attack of a thiolate on the 8-position of the flavin. Sever al lines of evidence indicate that the reaction takes place in the FAD binding site of the protein. The substrate specificity was investigat ed using amino acids with various lengths of side chain. L-Lysine and derivatives with similar side chain length are hydroxylated by lysine N6-hydroxylase. Ornithine, the lower homolog of lysine, was not hydrox ylated and did not affect the NADPH oxidase activity of the enzyme. On the other hand, homolysine accelerated the rate of NADPH oxidation bu t was not hydroxylated. Additional requirements for efficient hydroxyl ation were also investigated using a variety of substrate analogs. Fro m these studies a schematic structure of the active site of the enzyme was deduced. Sequence comparison of the FAD binding site of various f lavoproteins revealed possible factors for weak binding of the cofacto r in the case of lysine N6-hydroxylase.