Abstract
The vitamin K-dependent carboxylase is an integral membrane protein with five transmembrane domains (TMDs). It catalyzes the post-translational modification of specific glutamic acid residues of vitamin K-dependent proteins to gamma-carboxyglutamic acid residues. This posttranslational modification is critical for the biological functions of blood coagulation. The native enzyme is a single chain molecule with one disulfide bond. In this study, we have expressed carboxylase as two chains: residues 1–345 and 346–758 in the same insect cells. Our results show that these two fragments are assembled into a fully active enzyme and are joined by a disulfide. Affinity purification of the carboxylase C-terminal fragment (346–758) results in co-purification of the N-terminal fragment (1–345) even under reducing condition. This indicates that, in addition to the disulfide linkage between these two fragments, they are also linked by non-covalent interactions. One possibility is that the hydrophobic interactions between the TMDs play a role. According to carboxylase membrane topology, there are four TMDs (1–4) in the N-terminal fragment and one TMD (fifth) in the C-terminal fragment. The C-terminal fragment contains all glycosylation sites. When we introduced two prolines to disrupt the transmembrane helix in the wild type carboxylase’s fifth TMD, glycosylation was eliminated. This indicates that the domain is not inserted into the lumen of the ER, but remains in the cytoplasm. Therefore, as our results demonstrate, in the two chain carboxylase with its fifth TMD disrupted, the two chains do not form a disulfide bound nor do they associate through essential non-covalent TMD interactions. While proline residues can disrupt membrane helices as described above, they often occur at the interface between the membrane and the lumenal surface of ER; these prolines appear to affect the chain orientation as it exits the membrane. There is a proline at residue 378 near the lumenal surface of the fifth TMD helix of carboxylase. To examine P378’s effect on disulfide bond formation, we mutated it to leucine. Results show that less disulfide bond formed in the two chain mutant carboxylase and the protein was significantly degraded when compared to the unmutated two chain molecule. Based on our results, we conclude the following:
1) the two chain carboxylase is assembled into a single molecule in vivo and the two chains are joined by a disulfide bond, the enzyme carboxylates gla-containing substrates and binds propeptide with affinity similar to that of wild type enzyme. Therefore, this molecule is a good model for structural studies of TMD interactions and disulfide bond formation; 2) TMD association in the membrane is important for the orientation of the N- and C-terminal portions of carboxylase to be assembled into the active enzyme; 3) and finally proline residue 378 at the lumenal interface of the fifth TMD plays a key role in the conformation which promotes disulfide formation.
Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095
