Processive post-translational modification. Vitamin K-dependent carboxylation of a peptide substrate.
Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive post-translational modification (i.e. multiple carboxylations occur during a single association between enzyme and substrate). Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids -18 to 41 of the human blood clotting enzyme factor IX. Mass spectrometry was used to determine the number of gamma-carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction. Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times. This observation can only be explained by two types of mechanisms. In a processive mechanism, multiple carboxylations could occur during a single substrate binding event. Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S. Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translation modification.
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
D P
DP
Morris
R D
RD
Stevens
D J
DJ
Wright
D W
DW
Stafford
D000595:Amino Acid Sequence; D019735:Carbon-Carbon Ligases; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase