Mutations affecting the activity of urokinase-type plasminogen activator.
Mutagenesis throughout the single-chain urokinase-type plasminogen activator (scu-PA) cDNA molecule, followed by expression of the mutant genes and secretion of the resulting mutant proteins from yeast, has been used to determine the amino acid residues important for activity of scu-PA molecules. Twelve out of 13 colonies secreting variant scu-PA molecules with decreased ability to form a zone of fibrinolysis had mutant genes with a single codon alteration in the serine protease encoding domain (B-chain). Many of these changes are of highly conserved residues in the serine proteases and are consequently of considerable interest. A model three-dimensional structure of the protease domain of urokinase was used to explain the basis for the effects of these down mutations. The model showed that the strongest down mutations result from either interference of the mutated side chain with substrate binding at the active site or the introduction of bulky or charged groups at structurally sensitive internal positions in the molecule. Attempts to find second site revertants of five down mutants, altered either at the plasmin activation site or near the serine at the active site, only resulted in same-site revertants, with the original or closely related amino acids restored.
Davidow
L S
LS
Collaborative Research Inc., Waltham, MA 02154.
Dumais
D R
DR
Smyth
A P
AP
Greer
J
J
Moir
D T
DT
D001483:Base Sequence; D001665:Binding Sites; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D011498:Protein Precursors; D014568:Urokinase-Type Plasminogen Activator