Bovine liver aspartyl beta-hydroxylase. Purification and characterization.
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.
Wang
Q P
QP
Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
VanDusen
W J
WJ
Petroski
C J
CJ
Garsky
V M
VM
Stern
A M
AM
Friedman
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight
D001224:Aspartic Acid; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase