Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion.
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.
Anderson
M S
MS
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065, USA. matt_anderson@merck.com
Eveland
S S
SS
Onishi
H R
HR
Pompliano
D L
DL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D005982:Glutathione Transferase / Q000096:biosynthesis; D007700:Kinetics; D010453:Peptide Synthases / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D010453:Peptide Synthases; C060628:UDP-N-acetylmuramoylalanyl-D-glutamyllysine-D-alanyl-D-alanine ligase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate