title: A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains.
abstract: Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
authors.forename: D
authors.initials: D
authors.lastname: Bizub
authors.forename: I T
authors.initials: IT
authors.lastname: Weber
authors.forename: C E
authors.initials: CE
authors.lastname: Cameron
authors.forename: J P
authors.initials: JP
authors.lastname: Leis
authors.forename: A M
authors.initials: AM
authors.lastname: Skalka
mesh_terms: D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation
chemical_list: D010450:Endopeptidases