Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158.
The cDNA encoding full-length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced, and the recombinant scu-PA (rscu-PA) was expressed in Chinese hamster ovary cells. Two mutants, constructed by in vitro site-specific mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158) or to Glu158 (rscu-PA-Glu158), were also expressed in Chinese hamster ovary cells. Wild type and mutant rscu-PAs were purified to homogeneity by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration. The specific activity of the mutant scu-PAs on fibrin plates is very low (less than 1,000 IU/mg) compared to that of the wild type rscu-PA (44,000 IU/mg). The mutants, in contrast to the wild type rscu-PA, are not converted to amidolytically active two chain u-PA (tcu-PA) by plasmin and do not cause lysis of a 125I-fibrin-labeled plasma clot immersed in citrated plasma. However, in a purified system, both rscu-PA-Gly158 and rscu-PA-Glu158 activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (Km = 60-80 microM) but with a higher turnover rate constant (k2 = 0.01 s-1) as compared to the wild type rscu-PA (Km = 1.0 microM, k2 = 0.002 s-1). We conclude that conversion of scu-PA to tcu-PA is not a prerequisite for the activation of plasminogen. Substitution of Lys158 by Gly158 or Glu158 does, however, markedly decrease the stability of the Michaelis complex.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008239:Lysine; D009154:Mutation; D010957:Plasmids; D010960:Plasminogen Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D003062:Codon; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine