Journal article
Chemical Modification of Cysteine Residues Is a Misleading Indicator of Their Status as Active Site Residues in the Vitamin K-dependent γ-Glutamyl Carboxylation Reaction
The Journal of biological chemistry, Vol.279(52), pp.54079-54087
12/24/2004
DOI: 10.1074/jbc.M408945200
PMID: 15492002
Abstract
The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to γ-carboxy-glutamic acid by the integral membrane enzyme, γ-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys323 and Cys343. According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys323 and Cys343 may disrupt the three-dimensional structure, resulting in inactivation.
Details
- Title: Subtitle
- Chemical Modification of Cysteine Residues Is a Misleading Indicator of Their Status as Active Site Residues in the Vitamin K-dependent γ-Glutamyl Carboxylation Reaction
- Creators
- Jian-Ke Tie - University of North Carolina at Chapel HillDa-Yun Jin - University of North Carolina at Chapel HillDavid R. Loiselle - University of North Carolina at Chapel HillR. Marshall Pope - Invitrogen Corporation, Research and Development, Carlsbad, California 92008David L. Straight - University of North Carolina at Chapel HillDarrel W. Stafford - University of North Carolina at Chapel Hill
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.279(52), pp.54079-54087
- Publisher
- Elsevier Inc
- DOI
- 10.1074/jbc.M408945200
- PMID
- 15492002
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Language
- English
- Date published
- 12/24/2004
- Academic Unit
- Medicine Administration
- Record Identifier
- 9984627234302771
Metrics
9 Record Views