Processive post-translational modification. Vitamin K-dependent carboxylation of a peptide substrate

J Biol Chem. 1995 Dec 22;270(51):30491-8. doi: 10.1074/jbc.270.51.30491.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Carbon-Carbon Ligases*
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Escherichia coli
  • Glutamic Acid*
  • Humans
  • Kinetics
  • Ligases / isolation & purification
  • Ligases / metabolism*
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide Biosynthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Processing, Post-Translational*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Peptide Fragments
  • Peptides
  • Recombinant Proteins
  • Glutamic Acid
  • Ligases
  • Carbon-Carbon Ligases
  • glutamyl carboxylase