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[3H-R,S]EAL with high specific radioactivity to probe the timing of glutamyl Cgamma-3H cleavage relative to Cgamma-COO- bond formation by 14CO2 incorporation rates. Studies were conducted over a range of NaH14CO3 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 Cgamma-3H cleavage 11-13-fold and Cgamma-14CO2- formation 6-7-fold, converting a Cgamma-3H cleavage/Cgamma-14CO2- 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 Cgamma-3H cleavage from C-C bond formation. When the HCO3- concentration was varied, the V/K3H+/V/K14CO2 ratios rose as HCO3- fractional saturation dropped to a ratio of 9.3-10.8/l 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.
Li
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S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Furie
B C
BC
Furie
B
B
Walsh
C T
CT
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D002138:Calibration; D002245:Carbon Dioxide / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000378:metabolism; D019735:Carbon-Carbon Ligases; D002384:Catalysis; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D017693:Sodium Bicarbonate / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism*
D002250:Carbon Radioisotopes; D011498:Protein Precursors; D014316:Tritium; D014812:Vitamin K; D002245:Carbon Dioxide; D015055:1-Carboxyglutamic Acid; D017693:Sodium Bicarbonate; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase