Partial purification and characterization of bovine liver aspartyl beta-hydroxylase.
In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.
Gronke
R S
RS
Department of Pharmacology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Welsch
D J
DJ
VanDusen
W J
WJ
Garsky
V M
VM
Sardana
M K
MK
Stern
A M
AM
Friedman
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004220:Disulfides; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005164:Factor IX / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
D004220:Disulfides; D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D005164:Factor IX; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase; D010447:Peptide Hydrolases