PHE-LEU-GLU-GLU-LEU

Vitamin K-dependent carboxylation activity measured with pentapeptide substrate (Phe-Leu-Glu-Glu-Leu) gradually decreases upon in vivo injection of vitamin K to vitamin K-deficient rats. A decrease in pentapeptide carboxylation can also be observed by the in vitro addition of antibodies against prothrombin and other vitamin K-dependent proteins to the soluble system derived from vitamin K-deficient rat liver microsomes. In both cases, adding back in vitro partially decarboxylated vitamin K-dependent proteins or purified hepatic prothrombin precursor restores the level of pentapeptide carboxylation. After warfarin treatment, a 3-fold increase in carboxylation results, which can be abolished by giving cycloheximide along with the warfarin. However, the resulting decreased activity is restored by the in vitro addition of partially decarboxylated vitamin K-dependent proteins. These data are consistent with the hypothesis that (after warfarin treatment) increased peptide carboxylation is primarily due to activation of the system by precursor proteins, rather than synthesis of an increased amount of enzyme.

Vitamin K (2-methyl-3-phytyl-1,4-naphthoquinone) is required for the synthesis of prothrombin, Factor VII, Factor IX, Factor X, and a number of newly discovered proteins. These plasma proteins participate in calcium-dependent phospholipid membrane interactions which are mediated through the presence of gamma-carboxyglutamyl residues in their amino-terminal region. Vitamin K is required for the postribosomal conversion of glutamyl residues in liver precursors of these proteins to gamma-carboxyglutamyl residues in the completed plasma proteins. In the absence of vitamin K, or in the presence of vitamin K antagonists, animals produce plasma forms which lack the carboxylated residue. These proteins are nonfunctional because of their lack of phospholipid interaction. The vitamin K-dependent carboxylase which carries out this reaction has been studied in rat liver microsomal preparations where it will carboxylate the endogenous precursor proteins. Low-molecular-weight glutamyl-containing peptide substrates, such as Phe-Leu-Glu-Glu-Leu, which are homologous to regions of the prothrombin precursor, will also serve as substrates for the detergent-solubilized enzyme. This enzyme has been shown to require the reduced form of the vitamin and O2 but no ATP or a biotin-containing protein for its activity. The same microsomal preparations will also convert vitamin K to its 2,3-epoxide, and it is possible that activity may be related to the role of the vitamin in driving the carboxylase reaction.

Rationale: Traditionally, hydrogen/deuterium exchange (HDX) reactions are done in the solution phase. This usually involves incubating the protein with a suitable deuterating agent then acidifying the solution to quench the reaction. A more efficient method may be to conduct the reaction within the ion source of a mass spectrometer and subsequently analyze the products. Methods: Using the two electrospray emitters equipped on the Waters Synapt G1 mass spectrometer, HDX reactions were conducted within the ion source region in a controlled fashion ('dual-spray'). Peptide and protein solutions were electrosprayed through one emitter and the deuterating agent D2 O through the secondary electrospray emitter. For the relatively small peptides, Phe-Leu-Glu-Glu-Leu and oxytocin, the yield of products was calculated using deconvolution functions. Electrospray ionization (ESI) charge-state distributions and average number of deuterium exchanges were used to probe secondary and tertiary structures of ubiquitin, lysozyme, and cytochrome c in their native and unfolded states. Results: Clear shifts in isotope distributions indicated HDX occurring within the ion source. By ion mobility, simultaneous deuterium exchange for two isobaric species, the oxytocin monomer and dimer, was observed. For denatured ubiquitin, the 12+ and 13+ charge states have a lower average number of exchanges relative to the lower charge states which indicates that these charge states have segments which restrict the access of D2 O. Lysozyme has a linear relationship between the charge state and the average number of exchanges, indicating that lysozyme becomes increasingly unfolded as the charge state increases. The dual-spray HDX method was paired to high-performance liquid chromatography (HPLC) to demonstrate the applicability of the technique for probing gas-phase structures in protein mixtures. Conclusions: ESI droplets formed from a secondary emitter penetrate primary ESI droplets and change the solvent composition. Dual-spray HDX is demonstrated to be a more efficient method for probing the structure of proteins than solution-phase HDX since the acid quenching step can be surpassed.