Abstract
The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-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 Cys(323) and Cys(343). 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 Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.
Highlights
The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to ␥-carboxy-glutamic acid by the integral membrane enzyme, ␥-glutamyl carboxylase
Inactivation of Carboxylase by Thiol-reactive Reagents—Because vitamin K-dependent carboxylase is inactivated by a variety of thiol-reactive reagents, free cysteines have been implicated as comprising part of the carboxylase active site (3, 8 –10)
The hydrophobic maleimide derivative DABMI, which has a structure similar to AMS, does not inactivate carboxylase at concentrations up to 5 mM. These results suggest that cysteine residues that affect the enzyme activity by chemical modification may be located in the hydrophilic but not the hydrophobic regions of the carboxylase
Summary
Materials—All of the chemicals were reagent grade. AMS and DABMI were obtained from Molecular Probes (Eugene, OR). The mixtures were incubated on ice for 20 min, and carboxylase activity was determined using NaBH4-reduced VKH2. Identification of AMS-labeled Cysteine Residue of Carboxylase—Carboxylase or Na2S4O6-inactivated carboxylase were incubated with 1 mM AMS on ice for 20 min. The carboxylase bands were excised, and in-gel deglycosylation and trypsin digestion were performed as described previously [13, 19]. According to our previously published membrane topology [20], the C-terminal fragment with the last transmembrane domain of carboxylase should contain the HPC4 antibody recognition sequence, so we purified this fragment by affinity chromatography using the antibody column described before [20]. The active enzyme concentration was determined by fluorescence anisotropy titration of the enzyme against fluorescein-labeled consensus propeptide as described [18]
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