The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, c-carboxyglutamic acid, have been identified. To examine its biosynthesis of c-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin Kdependent epoxidase activity. The recombinant carboxylase has a molecular mass of 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues )18 to +10 of human proprothrombin and proFactor IX with K m values of 420 lM, 1.7 lM and 6 lM, respectively; the K m for vitamin K is 52 lM. The K m values for peptides based on the sequence of the conotoxin e-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 lM, 75 lM and 74 lM, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent c-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of c-carboxyglutamic acid synthesis in diverse biological systems.Keywords: blood coagulation; conotoxins; hemophilia; posttranslational processing; vitamin K.The vitamin K-dependent carboxylase catalyzes the posttranslational conversion of glutamic acid into c-carboxyglutamic acid in prothrombin, other blood coagulation proteins, and various vitamin K-dependent proteins [1,2]. In this reaction, CO 2 replaces the c-proton on specific glutamic acid residues of the peptide substrate to yield c-carboxyglutamic acid. This enzymatic reaction is unique in that it involves a strong base catalysis mechanism that requires a labile oxidized form of vitamin K [3]. The mammalian vitamin K-dependent carboxylase exhibits both carboxylase activity and vitamin K epoxidase activity [4]. Precursor proteins bearing within their propeptides the c-carboxylation-recognition site that binds directly to the carboxylase serve as substrates for this enzyme [5,6]. The recognition site is sufficient to direct c-carboxylation [7]. Cloning of the human and bovine carboxylases revealed a protein of 758 amino acids without obvious homology to other known proteins [8,9]. Cloning of other mammalian vitamin Kdependent carboxylases revealed marked (> 90%) aminoacid sequence co...

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.