3-13C-methionine-labelled E. coli alkaline phosphatase.

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Structure and Function

Background:

• Methionine residues are crucial for enzyme structure and function.
• Understanding enzyme conformational changes is vital in biochemistry.

Purpose of the Study:

• To investigate the structural environment of methionine residues in E. coli alkaline phosphatase.
• To explore the enzyme's conformational alterations using 13C-NMR spectroscopy.

Main Methods:

• Biosynthetic incorporation of 3-13C-methionine into E. coli alkaline phosphatase using a methionine-auxotrophic strain.
• 13C Nuclear Magnetic Resonance (NMR) spectroscopy analysis of the dimeric native enzyme.

Main Results:

• Resolution of at least five distinct methionine environments within the enzyme.
• Methionine chemical shifts indicate they are not in a free state.
• At acidic pH, distinct resonances suggest retained structure despite solvent exposure.

Conclusions:

• 3-13C-methionine labeled E. coli alkaline phosphatase serves as a valuable probe.
• The enzyme retains defined structural elements even under acidic conditions.
• This labeling approach facilitates the study of enzyme conformational dynamics.