Synthesis and biological properties of new hexapeptide substrates for vitamin K dependent carboxylase. Evidence for X-Pro cis/trans amide bond interconversions in prothrombin precursor fragment 18-23.

Three hexapeptide analogues, corresponding to sequence 18-23 of bovine prothrombin precursor [-Cys-Leu-Glu-Glu-Pro-Cys-] have been synthesized and evaluated as substrates for vitamin K dependent carboxylase. These new hexapeptides are moderately good substrates for the carboxylase but do not significantly inhibit carboxylation of Phe-Leu-Glu-Glu-Leu, a good substrate for the enzyme. Based on proton and carbon-13 NMR experiments, it is established that the conformation of sequence 18-23, which contains proline at position 22, has a trans amide bond for the Glu-Pro22 sequence in chloroform-d. This amide bond is converted to the cis amide geometry in Me2-SO-d6. It is proposed that good substrates for the carboxylase require a trans amide bond between residues 21 and 22.

Vitamin K-dependent carboxylase: synthesis of an inhibitor of the glutamyl binding site.

Liver microsomes contain a vitamin K and O2-dependent carboxylase that converts peptide-bound glutamyl residues to gamma-carboxyglutamate residues. The peptide Boc-O-phospho-Ser-O-phospho-Ser-Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe-Leu-Glu-Glu-Leu.

Synthesis of two analogs of a cyclic hexapeptide with disulfide bridge corresponding to bovine prothrombin precursor sequence 18-23. Extent of carboxylation by Vitamin K-Dependent carboxylase.

The synthesis of two analogs of sequence 18-23 of bovine prothrombin precursor is described. Hexapeptides Boc-Cys (Acm)-Leu-Glu(OBzl)-Glu(OBzl)-Pro-Cys (Acm)-OBzl and Ac-Cys(Acm-Leu-Glu(OBzl)-Glu(OBzl)-Pro-Cys(Acm)-OMe were synthesized in solution by stepwise addition of Boc-amino acids using dicyclohexylcarbodiimide/N-hydroxybenzotriazole as the coupling reagent. The acetamidomethyl groups were cleaved and oxidized, using iodine in methanol, to the protected cyclic disulfide in 62-69% yield. The O-benzyl groups were removed either by treatment with anhydrous hydrogen fluoride or hydrogen bromide in trifluoroacetic acid to give the cyclic hexapeptide disulfides, R1-Cys-Leu-Glu-Glu-Pro-Cys-OR2 where R1 - H or Ac and R2 = H or CH3. The cyclic hexapeptides were evaluated as substrates for vitamin K-dependent carboxylase. Both peptides are unusually poor substrates for the carboxylase, and each appears to inhibit carboxylation of Phe-Leu-Glu-Glu-Leu, a good substrate for the enzyme.

Synthesis of peptide analogues of prothrombin precursor sequence 5-9. Substrate specificity of vitamin K dependent carboxylase.

Thirty-five analogues of Phe-Leu-Glu-Glu-Leu, the pentapeptide sequence 5-9 of bovine prothrombin precursor, were synthesized and assayed as potential substrates or inhibitors of rat liver vitamin K dependent carboxylase. Carboxylation of substrate was determined by measuring the incorporation of carbon-24 labeled bicarbonate into product. Changes in substrate carboxylation produced by changing peptide chain length, amino acid chirality, or the distance separating the peptide chain backbone from the carboxyl group were measured. The data suggest that the carboxylase carboxylates L-glutamic acid residues and does not carboxylate L-aspartic acid, L-homoglutamic acid, glutamine, or D-glutamic acid residues; tri-through pentapeptides are better substrates than mono- or bis(amino acid) derivatives, and hydrophobic groups added to the N-terminus can produce better substrates for the enzyme. None of the synthetic substrates is carboxylated as effectively as the endogenous protein substrates for the enzyme. The effect of structure on additional parameters affecting carboxylation is discussed.