MULTI-SITE-SPECIFICITY OF THE VITAMIN-K-DEPENDENT CARBOXYLASE - IN-VITRO CARBOXYLATION OF DES-GAMMA-CARBOXYLATED BONE GLA PROTEIN AND DES-GAMMA-CARBOXYLATED PRO BONE GLA PROTEIN

The vitamin K-dependent carboxylase processes multiple glutamic acid r esidues to gamma-carboxyglutamic acid (Gla) residues in a limited numb er of proteins. The targeted proteins are synthesized with an amino-te rminal propeptide which has been shown to play an important role in ga mma-carboxylation, The specificity of the enzyme for each potential Gl a site, the direction of carboxylation, and the influence of a bound p ropeptide on these events are not understood, Des-gamma-carboxy forms of bone Gla protein (BGP), which contain potential Gla residues at pos itions 17, 21, and 24, were employed as model substrates to determine the multi-site-specificity of the enzyme. Recombinant bovine des-gamma -carboxylated proBGP (rdproBGP) and heat-decarboxylated BGP (dBGP), la cking a propeptide, were used as substrates for a bovine liver carboxy lase, and the in vitro reaction products were analyzed for the formati on of (CO2)-C-14 Gla, The di-Gla species was found to be the predomina nt product of in vitro carboxylation of both rdproBGP and dBGP at less than saturating concentrations of each substrate. Carboxylation of bo th substrates occurred preferentially at the more C-terminal potential Gla sites, residues 21 and 24. A similar pattern of carboxylation was observed with a rat bone cell carboxylase, suggesting no species or t issue variation in the enzyme specificity, Some tricarboxylated produc t accumulated during carboxylation of rdproBGP but not dBGP, suggestin g that the covalently bound propeptide directs more complete carboxyla tion of the Gla domain, in addition, monocarboxylated rdproBGP was fou nd to accumulate in the absence but not in the presence of a free nonc ovalently attached propeptide, indicating that free propeptide affects more efficient carboxylation of rdproBGP.