The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins.
The kinetics of activation of normal and gamma-carboxyglutamic acid (Gla)-deficient prothrombins isolated from cattle maintained for extended periods on the vitamin K antagonist dicoumarol were studied. The catalyst was prothrombinase, comprising isolated Factor Xa, Factor Va, phospholipid vesicles, and calcium ion. The Km and kcat values for prothrombins with 0, 1, 2, 5, 7, and 10 Gla residues were determined both by initial rate analysis and by integrated Michaelis-Menten-Henri analysis. Each of the Gla-deficient prothrombins exhibited kcat values similar to that of normal 10-Gla prothrombin but Km values that were 8- to 20-fold greater than that of the normal molecule. The increased Km coincided with a loss of Ca2+- and phospholipid-binding properties of the Gla-deficient prothrombins. The magnitude of the defect in both the kinetics of activation and Ca2+ and phospholipid binding is not progressive with the loss of Gla residues but rather appears abruptly with the loss of as few as 3 of the 10 Gla residues present in the normal substrate. The theoretical relationship between Km(app) and the dissociation constant (Kd) of the prothrombin-phospholipid interactions was derived. According to the result, the increase in apparent Km observed with the Gla-deficient prothrombins corresponds to at least a 100- to 1000-fold decrease in affinity for phospholipid compared to the affinity of normal prothrombin. In addition, the products of the activation of 10-Gla prothrombin were found to inhibit the activation of the Gla-deficient prothrombins.
Malhotra
O P
OP
Nesheim
M E
ME
Mann
K G
KG
D015055:1-Carboxyglutamic Acid / Q000172:deficiency*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003619:Dansyl Compounds / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005971:Glutamates / Q000172:deficiency*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*
D003619:Dansyl Compounds; D005971:Glutamates; D010446:Peptide Fragments; D010743:Phospholipids; D015055:1-Carboxyglutamic Acid; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D001728:Dicumarol; D011516:Prothrombin; D001120:Arginine; D002118:Calcium