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Biochemistry. 1995 Jul 25;34(29):9541-51.

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.

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  • 1Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706-1569, USA.


The vitamin K-dependent carboxylase processes multiple glutamic acid residues to gamma-carboxyglutamic acid (Gla) residues in a limited number of proteins. The targeted proteins are synthesized with an amino-terminal propeptide which has been shown to play an important role in gamma-carboxylation. The specificity of the enzyme for each potential Gla site, the direction of carboxylation, and the influence of a bound propeptide on these events are not understood. Des-gamma-carboxy forms of bone Gla protein (BGP), which contain potential Gla residues at positions 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), lacking a propeptide, were used as substrates for a bovine liver carboxylase, and the in vitro reaction products were analyzed for the formation of 14CO2 Gla. The di-Gla species was found to be the predominant product of in vitro carboxylation of both rdproBGP and dBGP at less than saturating concentrations of each substrate. Carboxylation of both 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 tissue variation in the enzyme specificity. Some tricarboxylated product accumulated during carboxylation of rdproBGP but not dBGP, suggesting that the covalently bound propeptide directs more complete carboxylation of the Gla domain. In addition, monocarboxylated rdproBGP was found to accumulate in the absence but not in the presence of a free noncovalently attached propeptide, indicating that free propeptide affects more efficient carboxylation of rdproBGP.

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