Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin.

The primary structure of rat liver xanthine dehydrogenase (EC 1.1.1.204) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 1,319 amino acid residues with a calculated molecular mass of 145,034 Da, including initiation methionine, and is homologous to the previously reported Drosophila melanogaster enzyme (Lee, C. S., Curtis, D., McCarron, M., Love, C., Gray, M., Bender, W., and Chovnick, A. (1987) Genetics 116, 55-66; Keith, T. P., Riley, M. A., Kreitman, M., Lewontin, R. C., Curtis, D., and Chambers, G. (1987) Genetics 116, 67-73) with an identity of 52%. The enzyme exists originally as the NAD-dependent type in a freshly prepared sample. When the purified NAD-dependent type enzyme was digested with trypsin, it cleaved into three fragments with molecular masses of 20, 40, and 85 kDa and was irreversibly converted to the O2-dependent type. Comparison of the amino-terminal sequences of the three peptide fragments with the cDNA-deduced sequence reveals that the 20-, 40-, and 85-kDa peptide fragments correspond residues to 1-184, 185-539, and 540-1319 of the enzyme, respectively. Comparison of the 5'-p-fluorosulfonylbenzoyladenosine-labeled peptide sequence of the chicken enzyme (Nishino, T., and Nishino, T. (1989) J. Biol. Chem. 264, 5468-5473) reveals that the NAD binding site is associated with the 40-kDa fragment portion of the enzyme. Hydropathy analysis around the cysteine residues suggests that the 2Fe/2S sites are associated with the 20-kDa fragment portion of the enzyme.