EXPRESSION IN ESCHERICHIA-COLI AND REFOLDING OF THE MALONYL- ACETYLTRANSFERASE DOMAIN OF THE MULTIFUNCTIONAL ANIMAL FATTY-ACID SYNTHASE/

A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant p rotein refolded in vitro as a catalytically active malonyl-/acetyltran sferase. Kinetic properties of the refolded recombinant enzyme were in distinguishable from those of a transferase preparation derived from t he natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independ ent protein, Replacement of the active site Ser-581 (full length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibi tion was afforded by both malonyl- and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocar bonate. The rate of acylation of the active site serine residue was al so reduced by several orders of magnitude in the His-683 --> Ala mutan t. These results indicate that His-683 plays an essential role in cata lysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity.