KINETIC MECHANISM OF THE ESCHERICHIA-COLI UDPMURNAC-TRIPEPTIDE D-ALANYL-D-ALANINE-ADDING ENZYME - USE OF A GLUTATHIONE-S-TRANSFERASE FUSION

The D-alnyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of amy]-L-gamma-D-Glu-meso-diaminopimelyl -D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escher ichia coli and expressed as a glutathione S-transferase (GST) fusion u sing the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one s tep by affinity chromatography. The purified fusion protein was strong ly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered b y changes in assay pH or by cleavage from the fusion partner. However, this effect was suppressed by the addition of 0.5 M NaCl. Initial vel ocity and dead-end inhibitor studies with the fusion enzyme were most consistent with a sequential ordered kinetic mechanism for the forward reaction in which ATP binds to free enzyme, followed by tripeptide an d D-Ala-D-Ala in sequence prior to product release. Reported homologie s between the MurF protein and the three preceding steps of cytoplasmi c murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these en zymes will be found to proceed via this mechanism.