Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin.
An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.
Pei
G
G
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Baker
K
K
Emfinger
S M
SM
Fowlkes
D M
DM
Lentz
B R
BR
D015055:1-Carboxyglutamic Acid / Q000032:analysis; D000409:Alanine / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D009154:Mutation; D010957:Plasmids; D011516:Prothrombin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012694:Serine / Q000235:genetics*; D014757:Viper Venoms
D011994:Recombinant Proteins; D014757:Viper Venoms; D012694:Serine; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D004247:DNA; D010450:Endopeptidases; C020097:ecarin; D000409:Alanine