Bovine liver aspartyl beta-hydroxylase. Purification and characterization

• Published in: The Journal of biological chemistry 1991-07, Vol.266 (21), p.14004-14010
• Main Authors: QINGPING WANG, VANDUSEN, W. J, PETROSKI, C. J, GARSKY, V. M, STERN, A. M, FRIEDMAN, P. A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Aspartic Acid - metabolism; Biological and medical sciences; Blotting, Western; Cattle; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; liver; Liver - enzymology; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - immunology; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Mixed Function Oxygenases - ultrastructure; Molecular Sequence Data; Molecular Weight; Oxidoreductases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92802-4

The alpha-ketoglutarate-dependent dioxygenase, L-asp(L-Asn)-beta-hydroxylase which posttranslationally hydroxylates specific aspartic acid (asparagine) residues within epidermal growth factor-like domains was purified from bovine liver and characterized. A 52-kDa and a 56-kDa species of this enzyme, which accounted for 60 and 30% of the total enzymatic activity, respectively, were purified to apparent homogeneity. Amino-terminal sequence analyses and immunoblots utilizing antisera raised to the intact 52-kDa species as well as to two complementary fragments of this species demonstrated that the 52- and 56-kDa species differ by a 22-amino acid amino-terminal extension. The remaining 10% of the purified enzymatic activity could be accounted for by the presence of immunologically related higher molecular mass forms (56-90 kDa) of L-Asp(L-Asn)-beta-hydroxylase. Strong evidence was obtained from the results of immunoextraction studies that L-Asp(L-Asn)-beta-hydroxylase can be identified with the purified proteins. Kinetic and physical studies suggest that L-Asp(L-Asn)-beta-hydroxylase exists as a monomer with a compact catalytic domain and an extended protease-sensitive amino terminus whose function remains to be determined. Since the purified L-Asp(L-Asn)-beta-hydroxylase hydroxylated both L-Asp- and L-Asn-containing substrates, it is possible that a single enzyme is responsible for the hydroxylation of Asp and Asn residues in vivo.