Hydroxyproline 2-epimerase of Pseudomonas. Subunit structure and active site studies

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Hydroxyproline 2-epimerase from Pseudomonas putida is crucial for amino acid metabolism.
• Understanding enzyme active sites is key to elucidating catalytic mechanisms.

Purpose of the Study:

• To purify and characterize hydroxyproline 2-epimerase from Pseudomonas putida.
• To investigate the active site reactivity and subunit stoichiometry of the enzyme.

Main Methods:

• Enzyme purification to homogeneity.
• Active site alkylation with labeled and unlabeled reagents.
• Tryptic hydrolysis and peptide sequencing.
• Analysis of enzyme subunit composition.

Main Results:

• Purified hydroxyproline 2-epimerase consists of two identical subunits.
• Enzyme alkylation demonstrated half-site reactivity, with only one active site incorporating an alkyl group.
• Sequencing of the active site peptide revealed homology with clostridial proline racemase.

Conclusions:

• Pseudomonas putida hydroxyproline 2-epimerase exhibits half-site reactivity.
• The enzyme's active site structure and function share similarities with proline racemase.