PMID:8973200
| Citation |
Anderson, MS, Eveland, SS, Onishi, HR and Pompliano, DL (1996) Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion. Biochemistry 35:16264-9 |
|---|---|
| Abstract |
The D-alanyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escherichia coli and expressed as a glutathione S-transferase (GST) fusion using the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one step by affinity chromatography. The purified fusion protein was strongly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered by 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 velocity 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 and D-Ala-D-Ala in sequence prior to product release. Reported homologies between the MurF protein and the three preceding steps of cytoplasmic murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these enzymes will be found to proceed via this mechanism. |
| Links |
PubMed Online version:10.1021/bi961872+ |
| Keywords |
Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Base Sequence; Cloning, Molecular; DNA Primers; Escherichia coli/enzymology; Glutathione Transferase/biosynthesis; Kinetics; Peptide Synthases/biosynthesis; Peptide Synthases/metabolism; Polymerase Chain Reaction; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/metabolism; Substrate Specificity |
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