Biotin protein ligase from Saccharomyces cerevisiae - The N-terminal domain is required for complete activity

Catalytically active biotin protein ligase from Saccharomyces cerevisiae (E C 6.3.4.15) was overexpressed in Escherichia coil and purified to near homo geneity in three steps. Kinetic analysis demonstrated that the substrates A TP, biotin, and the biotin-accepting protein bind in an ordered manner in t he reaction mechanism. Treatment with any of three proteases of differing s pecificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an expose d linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa doma in containing the active site, The protease susceptibility of this linker r egion was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates, Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein Ligase activity, In vitro assays performed wit h purified N-terminally truncated enzyme revealed that removal of the N-ter minal domain reduced BPL activity by greater than 3500-fold, Our data indic ate that both the N-terminal domain and the C-terminal domain containing th e active site are necessary for complete catalytic function.