N-bromoacetyl-peptide substrate affinity labeling of vitamin K dependent carboxylase.
Vitamin K dependent carboxylase (carboxylase) is a membrane-associated endoplasmic reticular enzyme that catalyzes the conversion of certain glutamate residues of essential blood coagulation proteins to gamma-carboxyglutamyl (Gla) residues. A series of N-bromoacetyl-peptide substrate affinity labels based on the Gla domain of these blood-clotting proteins was synthesized, and the substrate and inactivator kinetic parameters were assessed. The most promising of these affinity peptides, N-bromoacetyl-FLEELY, was both substrate for carboxylase and an irreversible time-dependent inactivator of the enzyme, inactivating 80% of carboxylase under pseudo-first-order conditions. Addition of saturating amounts of a competing peptide substrate completely abolished the inhibitory properties of N-bromoacetyl-FLEELY, consistent with inactivation occurring at the active site. The partition ratio of inactivation/carboxylation was 1/30. The 94-kDa carboxylase was purified to 15-50% purity by a modification of a recent protocol [Wu, S.-M., Morris, D. P., & Stafford, D. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2236-2240] and covalently labeled with N-bromoacetyl-FLEEL[125I]Y. On silver-stained 10% sodium dodecyl sulfate-polyacrylamide gels, the predominant radiolabeled band was the 94,000 molecular weight species. This result independently validates that the 94-kDa protein is a carboxylase.
Kuliopulos
A
A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Cieurzo
C E
CE
Furie
B
B
Furie
B C
BC
Walsh
C T
CT
D000085:Acetates / Q000494:pharmacology; D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D019735:Carbon-Carbon Ligases; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
D000085:Acetates; D000345:Affinity Labels; D009842:Oligopeptides; C077181:N-bromoacetyl-phenylalanyl-leucyl-glutamyl-glutamyl-leucyl-tyrosine; C016274:bromoacetate; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase