Journal article
Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
The Journal of biological chemistry, Vol.278(46), pp.45468-45475
11/14/2003
DOI: 10.1074/jbc.M309164200
PMID: 12963724
Abstract
Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033-13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.
Details
- Title: Subtitle
- Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- Creators
- Jian-Ke Tie - Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USAVasantha P MutucumaranaDavid L StraightKevin L Carrick - University of North Carolina at Chapel HillR Marshall Pope - University of North Carolina at Chapel HillDarrel W Stafford
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.278(46), pp.45468-45475
- DOI
- 10.1074/jbc.M309164200
- PMID
- 12963724
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Grant note
- HL48318 / NHLBI NIH HHS
- Language
- English
- Date published
- 11/14/2003
- Academic Unit
- Medicine Administration
- Record Identifier
- 9984627292902771
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