The propeptide of the vitamin K-dependent carboxylase substrate accelerates formation of the gamma-glutamyl carbanion intermediate

Vitamin K-dependent carboxylase catalyzes the post-translational gamma-carboxylation of 9-12 glutamyl residues of several blood coagulation proteins. Carboxylase purified from Chinese hamster ovary (CHO) cells as a recombinant FLAG-carboxylase fusion protein [Sugiura, I., et al. (1996) J. Biol. Chem. 271, 17837-17844] was utilized with pentapeptide substrate FL[H-3-R,S]EAL with high specific radioactivity to probe the timing of glutamyl C-gamma-H-3 cleavage relative to C-gamma-COO- bond formation by (CO2)-C-14 incorporation rates. Studies were conducted over a range of (NaHCO3)-C-14 concentrations to assess uncoupling of gamma-glutamyl carbanion formation and over a range of concentrations of ProPT18, the 18-residue peptide corresponding to the -18 to -1 propeptide region of prothrombin known to affect the catalytic efficiency of carboxylase. At saturation, ProPT18 accelerates C-gamma-H-3 cleavage 11-13-fold and C-gamma-(CO2-)-C-14 formation 6-7-fold, converting a C-gamma-H-3 cleavage/C-gamma-(CO2-)-C-14 formation ratio of 1.2-1.4 in the absence of ProPT18 to 2.3-2.8 in its presence, a relative increase in and uncoupling of C-gamma-H-3 cleavage from C-C bond formation. When the HCO3- concentration was varied, the V/K3H+/V/K-14CO2 ratios rose as HCO3- fractional saturation dropped to a ratio of 9.3-10.8/1 at low bicarbonate, indicating an uncoupling of nine out of ten gamma-glutamyl carbanion formations from carboxylative capture, consistent with prior reports on microsomal enzyme [Larson, A. E., et al. (1981) J. Biol. Chem. 256, 11032-11035]. These results with pentapeptide substrate FLEAL validate reversible gamma-glutamyl carbanion formation by pure carboxylase and indicate the ProPT18 increase in catalytic efficiency is in selective lowering of an energy barrier preceding the gamma-glutamyl carbanion intermediate.