The vitamin K-dependent carboxylation reaction

Summary Gammacarboxyglutamic acid (Gla) is an abnormal amino acid, which occurs in a number of proteins. It was discovered about 10 years ago in the four vitamin K-dependent blood clotting factors and it could be demonstrated that Gla is formed in a post-translational modification step, which requires a carboxylating enzyme system (carboxylase) and vitamin K. Since at the time of this discovery the earlier mentioned clotting factors were the only proteins known to be synthesized in a vitamin K-dependent way, it has been assumed for many years that the blood clotting system was unique in this respect. Recently it has been demonstrated, however, that vitamin K-dependent carboxylase is not restricted to the liver (the place of synthesis of the clotting factors) but that it is also present in other tissues such as lung, kidney, spleen and testis. Moreover, numerous Gla-containing proteins have been detected, although in most cases their function is not wholly understood. It seems that (like for instance the glycosylation) the vitamin K-dependent carboxylation is a normal post-translational. modification, which is required for the correct function of a certain class of Ca²⁺-binding proteins.     

Soluble enzyme system for vitamin K-dependent carboxylation.

The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.     

Oxygen requirements for vitamin K-dependent carboxylation and epoxide formation

The requirement of vitamin K-dependent carboxylation for oxygen was determined. Carboxylation was not detected at oxygen concentrations less than 0.05 mM or in the absence of vitamin K epoxide formation. Epoxide formation was detectable at 0.05 mM and was maximal at 0.10 mM O2. Carboxylation increased with oxygen concentrations over the range of 0.10 to 0.25 mM. At oxygen concentrations at which epoxide formation was maximal, the ratio of epoxide formation to carboxylated product was approx. 3.5:1. The data are consistent with the hypothesis that an oxygenated vitamin K intermediate is required for carboxylation.     

Stimulation of vitamin K-dependent carboxylation by pyridoxal-5'-phosphate.

This paper presents evidence that the approximately two-fold increase in vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluLeu, but not of endogenous protein substrate, brought about by pyridoxal-5′-phosphate, is due to binding of the pyridoxal-5′-phosphate to microsomal enzyme(s), rather than to the pentapeptide. Pyridoxine inhibits this peptide carboxylation, while pyridoxal, pyridoxamine, and pyridoxamine-5′-phosphate have no effect on the reaction.     

Bimodal frequency distribution of rat hepatic vitamin K-dependent carboxylation rate

The time course of vitamin K-dependent carboxylation was studied in an in vitro rat hepatic microsomal system. This method is based on incorporation of radiolabelled CO2 into endogenous substrate proteins. Forty rats were studied in order to characterize the intrinsic formation rate (V/KM) of carboxylated vitamin K-dependent proteins and the maximum amount of endogenous substrate available for vitamin K-dependent carboxylation (P infinity; normalized for the total amount of microsomal protein harvested). The frequency distributions of V/KM and P infinity values were both well described as the sum of two Gaussian components, each representing about 40% and 60% of the populations.     

Nature of products formed in the vitamin K-dependent carboxylation of synthetic peptides

Vitamin K-dependent carboxylation of synthetic Phe-Leu-Glu-Glu-Val by rat liver microsomes yields a secondary product, which has been identified as Leu-Gla-Glu-Val. Similar results are obtained with other synthetic substrates. The microsomal preparation has been shown to contain an aminopeptidase activity which splits carboxylation products and substrates but is unable to hydrolyse the Leu-Gla peptide bond.     

Mechanism of action of t-butyl hydroperoxide in the inhibition of vitamin K-dependent carboxylation

t-Butyl hydroperoxide has been studied as a possible competitive inhibitor of the vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluIle. Under standard carboxylating conditions the concentrations of reduced phylloquinone and phylloquinone were followed by high-pressure liquid chromatography during 30-min incubations of Triton-solubilized microsomes from rat liver. Under these conditions supporting linear rates of carbon dioxide fixation for 20–30 min, the vitamin KH₂ concentration decreased exponentially to less than 5% of its initial value in 30 min principally due to autooxidation. In the presence of 10 mm t-butyl-OOH, however, the oxidation of vitamin KH₂ was greatly accelerated with none being detected after 7 min. In general, the rate of carboxylation of peptide paralleled the KH₂ concentration. After cessation of carboxylation in the presence of t-butyl-OOH the readdition of KH₂ stimulated additional ¹⁴CO₂ fixation. A known competitive inhibitor of vitamin K, 2-chlorophylloquinone, did not accelerate the oxidation of KH₂ but nonetheless inhibited the vitamin K-dependent carboxylation in a competitive manner. These data have led us to conclude that t-butyl-OOH is not a competitive inhibitor of the vitamin K-dependent carboxylase at the active site of the enzyme but merely acts to promote the oxidation of KH₂.     

Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase

The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-Leu-Glu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnCl2 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation's ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.     

Fate of the activated gamma-carbon-hydrogen bond in the uncoupled vitamin K-dependent gamma-glutamyl carboxylation reaction

The gamma-glutamyl carboxylation reaction proceeds by an initial vitamin K-dependent gamma-C-H glutamyl bond cleavage and a subsequent carboxylation of the activated glutamyl residue. This system is easily uncoupled such that at low CO2 concentrations which limit the extent of carboxylation there is no effect on the rate of C-H bond cleavage. In an uncoupled system, the fate of activated glutamyl residues is to incorporate a hydrogen as demonstrated by the recovery of only unaltered glutamyl residues from digests of uncoupled reactions. In addition, in reactions carried out in tritiated, deuterated water mixtures, tritium is incorporated into the gamma positions of the glutamyl residues of peptide substrates in a vitamin K-dependent process, indicating that the hydrogen incorporated must ultimately come from solvent. These results, while not proof, put severe restraints on a radical mechanism while favoring a carbanion mechanism.     

Vitamin K-dependent carboxylation of poly-L-glutamate.

Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length.     

Evidence for vitamin K semiquinone as the functional form of vitamin K in the liver vitamin K-dependent protein carboxylation reaction.

The protein carboxylating system derived from vitamin K-deficient rat liver microsomes functions in detergent solution if vitamin K₁, NADH, dithiothreitol, CO₂ and O₂ are added. The requirements for added NADH, dithiothreitol and O₂ are all eliminated by the use of vitamin K₁ hydroquinone in place of quinone. The use of the hydroquinone gives a more rapid reaction and a higher yield than does the quinone plus reducing system. The reaction proceeding from either the vitamin K₁ quinone or hydroquinone is blocked by the spin-trapping agent, 5,5-dimethyl-l-pyrroline-N-oxide, suggesting that the active form of vitamin K is the semiquinone.     

Vitamin K-dependent carboxylation of blood coagulation proteins

Prothrombin is converted from an inactive precursor to a biologically active protein by vitamin K-dependent carboxylation of ten glutamic acid residues in the precursor.     

Synthesis of diastereoisomeric peptides incorporating cycloglutamic acids. Substrate specificity of vitamin K-dependent carboxylation.

Tripeptides Boc-X-Glu-Val where X is alpha-methyl glutamic acid or various cyclic analogues of glutamic acid, such as 1-amino-1,3-dicarboxycyclohexane (cis or trans-CHGA) or -cyclopentane (cis or trans-CPGA) have been synthesized. Methods for the selective protection, activation, and coupling of such unnatural amino acids are described. The peptides, which are potential competitive inhibitors of the vitamin K-dependent carboxylation, have been preliminarily tested with the rat liver microsomal carboxylase and found to be effective substrates of the carboxylation reaction.     

A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3- into prothrombin.

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.     

Evidence for the involvement of superoxide in vitamin K-dependent carboxylation of glutamic acid residues of prothrombin.

The formation of vitamin K epoxide and the vitamin K-dependent carboxylation of glutamic acid residues present in synthetic substrates and decarboxyprothrombin are both inhibited by superoxide dismutase. Catalase only inhibits the generation of vitamin K epoxide, suggesting that the carboxylation and epoxidation reactions are not inter-dependent.