PMID:8075064
| Citation |
Brown, ED, Marquardt, JL, Lee, JP, Walsh, CT and Anderson, KS (1994) Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ. Biochemistry 33:10638-45 |
|---|---|
| Abstract |
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) |
| Links | |
| Keywords |
Alkyl and Aryl Transferases; Bacterial Proteins/metabolism; Binding Sites; Catalysis; Escherichia coli/enzymology; Kinetics; Magnetic Resonance Spectroscopy; Phosphoenolpyruvate/metabolism; Transferases/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.
