Evidence of kinetic control of ligand binding and staged product release in MurA (enolpyruvyl UDP-GlcNAc synthase)-catalyzed reactions .
MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.
Jackson
Sean G
SG
Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton,Ontario L8S 4M1, Canada.
Zhang
Fuzhong
F
Chindemi
Paul
P
Junop
Murray S
MS
Berti
Paul J
PJ
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D005578:Fosfomycin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D029968:Escherichia coli Proteins; D008024:Ligands; D005578:Fosfomycin; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase; D003545:Cysteine