Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ.
The MurZ enzyme catalyzes enolpyruvoyl transfer from phosphoenolpyruvate to the 3-OH of UDP-N-acetylglucosamine (UDP-GlcNAc) in Escherichia coli peptidoglycan biosynthesis. The kinetic mechanism of MurZ has been shown to involve the generation of a non-covalently bound tetrahedral phospholactoyl-UDP-GlcNAc intermediate [Marquardt, J.L., et al. (1993) J. Am. Chem. Soc. 115, 10398-10399]. In the work described here, MurZ overproduced in E. coli copurified with 1 equiv of bound PEP. Enzyme free of bound PEP was prepared by incubation of purified MurZ with UDP-GlcNAc followed by removal of small molecules. Addition of either [14C]PEP or [32P]PEP to PEP-free enzyme led to stoichiometric labeling. The MurZ-PEP complex was stable to 6.7 M urea, and digestion of the [32P]PEP-labeled protein followed by SDS-PAGE generated a labeled peptide of molecular weight 5000. Solution NMR of MurZ incubated with [2-13C]PEP suggested a tetrahedral phospholactoyl enzyme adduct attached via C-2 to an enzyme nucleophile. The kon for generation of the phospholactoyl enzyme in the absence of UDP-GlcNAc was 0.24 microM-1 s-1, too slow to represent the binding order of the kinetically preferred pathway since the lower limit of the second-order binding constant for PEP (kcat/Km) is 15 microM-1 s-1. Rapid chemical quench analysis under single-turnover conditions using [32P]PEP in the presence of UDP-GlcNAc demonstrated that the covalent enzyme-phospholactoyl adduct appeared and decayed on a time scale consistent with catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Brown
E D
ED
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Marquardt
J L
JL
Lee
J P
JP
Walsh
C T
CT
Anderson
K S
KS
D019883:Alkyl and Aryl Transferases; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010728:Phosphoenolpyruvate / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
D001426:Bacterial Proteins; D010728:Phosphoenolpyruvate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase