The active site of membrane-bound meizothrombin. A fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va.
The distance between the phospholipid surface and the active site of membrane-bound meizothrombin, a derivative of prothrombin, was determined directly using fluorescence energy transfer. The active site of prothrombin was exposed after a single cleavage by Echis carinatus protease in the presence of [5-(dimethylamino)-1-naphthalenesulfonyl]glutamylglycylarginyl+ ++ (DEGR) chloromethyl ketone to yield DEGR-meizothrombin and thereby minimize secondary proteolysis. When DEGR-meizothrombin was titrated with 80% phosphatidylcholine, 20% phosphatidylserine vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes in the active sites of the membrane-bound proteins and the acceptor dyes at the outer surface of the phospholipid bilayer. This energy transfer required both Ca2+ and phosphatidylserine. Assuming k2 = 2/3, the dependence of the efficiency of energy transfer upon the acceptor density showed that the distance of closest approach between the active site probe and the bilayer surface was 71 +/- 2 A. In the presence of factor Va, the distance was 67 +/- 3 A. These direct measurements show that the active site of meizothrombin is located far above the membrane surface. Also, association of factor Va with meizothrombin on the phospholipid surface appears to cause a slight movement of the meizothrombin protease domain toward the membrane surface. The environment of the dansyl dye covalently attached to the active site of meizothrombin was particularly sensitive to the presence of calcium: addition of Ca2+ ions to metal-free DEGR-meizothrombin reduced the dansyl fluorescence lifetime from 11.7 to 9.0 ns and the dansyl emission intensity by 24%. Hence, the conformation of the active site changed when Ca2+ ions bound to meizothrombin. Since the intensity change was half-maximal at 0.2 mM and was also elicited by the binding of Mg2+ ions, this spectral change correlates with the calcium-dependent conformational change previously observed in fragment 1. We conclude, therefore, that the binding of Ca2+ ions to meizothrombin and, by extension, perhaps to prothrombin, elicits a conformational change that extends beyond the fragment 1 domains into the distant (cf. above) active site or protease domain. The association of factor Va with membrane-bound DEGR-meizothrombin increased both the dansyl emission intensity (by 7%) and polarization. This intensity change and the factor-Va dependent change in energy transfer indicate that the cofactor of the prothrombinase complex functions to modulate the conformation and orientation of both the substrate and the enzyme of the complex.
Armstrong
S A
SA
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Husten
E J
EJ
Esmon
C T
CT
Johnson
A E
AE
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D004792:Enzyme Precursors / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D015943:Factor Va / Q000494:pharmacology*; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin
D002413:Cations, Divalent; D004792:Enzyme Precursors; D008081:Liposomes; D008563:Membrane Lipids; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; D015943:Factor Va; D004950:Esterases; D010447:Peptide Hydrolases; D014357:Trypsin; D013917:Thrombin; C026812:meizothrombin; D002118:Calcium