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The Kinetic Characterization of Escherichia c oli MurG Using Synthetic Substrate Analogues

1999; American Chemical Society; Volume: 121; Issue: 37 Linguagem: Inglês

10.1021/ja991556t

1943-2984

Sha Ha, Emmanuel J. Chang, Mei-Chu Lo, Hongbin Men, Peter J. Park, Min Ge, Suzanne Walker,

Biochemical and Molecular Research

Bacterial resistance to existing antibiotics poses a serious threat to human health. Because the peptidoglycan layer surrounding bacterial cells is essential for survival, the enzymes involved in peptidoglycan biosynthesis are attractive targets for the design of new antibiotics. Unfortunately, many of these enzymes are difficult to study because substrates to monitor enzymatic activity are either not available or not soluble under suitable assay conditions. These problems can be solved by utilizing synthetic alternative substrates. We recently reported the synthesis of a soluble substrate analogue for MurG, the enzyme that forms the β-(1,4)-N-acetylglucosaminyl-N-acetylmuramyl pentapeptide subunit of peptidoglycan. Using this substrate analogue, we have been able to develop a direct assay to monitor the activity of the enzyme. We now report the purification of Escherichia coli MurG and information on its kinetic properties and substrate requirements in the absence of membranes. This work lays the foundation for detailed mechanistic and structural investigations of this essential bacterial enzyme.