Kinetic Mechanism of the Escherichia coli UDPMurNAc-Tripeptide D-ALANYL-D-alanine-Adding Enzyme: Use of a Glutathione S-Transferase Fusion
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- 作者：Matt S. Anderson ; Suzanne S. Eveland ; H. Russell Onishi ; and David L. Pompliano
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 17, 1996
- 年：1996
- 卷：35
- 期：50
- 页码：16264 - 16269
- 全文大小：363K
- 年卷期：v.35,no.50(December 17, 1996)
- ISSN：1520-4995
文摘
The D-alanyl-D-alanine-adding enzymeencoded by the murF gene catalyzes theATP-dependentformation ofUDP-N-acetylmuramyl-L-
-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala(UDP-MurNAc-tripeptide). MurF has been cloned from Escherichiacoli and expressed as a glutathione S-transferase(GST)fusion using the tac promoter-based pGEX-KT vector.From induced, broken cell preparations, highlyactive fusion was recovered and purified in one step by affinitychromatography. The purified fusionprotein was strongly inhibited by substrate UDPMurNAc-tripeptide, aresponse unaltered by changes inassay pH or by cleavage from the fusion partner. However, thiseffect was suppressed by the addition of0.5 M NaCl. Initial velocity and dead-end inhibitor studies withthe fusion enzyme were most consistentwith a sequential ordered kinetic mechanism for the forward reaction inwhich ATP binds to free enzyme,followed by tripeptide and D-Ala-D-Ala insequence prior to product release. Reported homologiesbetweenthe MurF protein and the three preceding steps of cytoplasmic mureinbiosynthesis, MurC, -D, and -E,[Ikeda et al. (1990) J. Gen. Appl. Microbiol.36, 179-187], raise the prospect that all of theseenzymeswill be found to proceed via this mechanism.
-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala(UDP-MurNAc-tripeptide). MurF has been cloned from Escherichiacoli and expressed as a glutathione S-transferase(GST)fusion using the tac promoter-based pGEX-KT vector.From induced, broken cell preparations, highlyactive fusion was recovered and purified in one step by affinitychromatography. The purified fusionprotein was strongly inhibited by substrate UDPMurNAc-tripeptide, aresponse unaltered by changes inassay pH or by cleavage from the fusion partner. However, thiseffect was suppressed by the addition of0.5 M NaCl. Initial velocity and dead-end inhibitor studies withthe fusion enzyme were most consistentwith a sequential ordered kinetic mechanism for the forward reaction inwhich ATP binds to free enzyme,followed by tripeptide and D-Ala-D-Ala insequence prior to product release. Reported homologiesbetweenthe MurF protein and the three preceding steps of cytoplasmic mureinbiosynthesis, MurC, -D, and -E,[Ikeda et al. (1990) J. Gen. Appl. Microbiol.36, 179-187], raise the prospect that all of theseenzymeswill be found to proceed via this mechanism.