Relocating the active site of activated protein C eliminates the need for its protein S cofactor - A fluorescence resonance energy transfer study

The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane- bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa(2) = 2/3) between a f luorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 Angstrom, compared with 94 Angstrom for wildtype AP C, The gamma-carboxyglutamic acid domain substitution therefore lowered and /or reoriented the active site, repositioning it close to the 84 Angstrom o bserved for the APC protein S complex. Protein S enhances wild-type APC cle avage of factor Va at Arg(306), but the inactivation rate of factor Va Leid en by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC protein S complex are equivalent because the active site of the chimer ic protein is already positioned near the optimal location above the membra ne surface to cleave Arg(306). Thus, one mechanism by which protein S regul ates APC activity is by relocating its active site to the proper position a bove the membrane surface to optimize factor Va cleavage.