Hydroxyproline-2-epimerase of Pseudomonas: active-site peptides

Area of Science:

• Biochemistry
• Enzymology

Background:

• Hydroxyproline-2-epimerase is vital for collagen synthesis.
• Understanding enzyme active sites is key to drug development.

Purpose of the Study:

• To investigate the active site of hydroxyproline-2-epimerase.
• To identify the amino acid residue modified by iodoacetate.

Main Methods:

• Enzyme inactivation using 14C-iodoacetate.
• Substrate protection assays.
• Tryptic digestion and peptide isolation via chromatography-electrophoresis.

Main Results:

• Complete enzyme inactivation correlated with iodoacetate incorporation.
• Substrate presence prevented inactivation and incorporation.
• Two radioactive tryptic peptides were isolated, with radioactivity in a 2:1 ratio.
• Peptides contained similar compositions, suggesting lysine and arginine residues, and an N-terminal cysteine.

Conclusions:

• A cysteine residue is located at or near the active site of hydroxyproline-2-epimerase.
• This cysteine is likely involved in substrate binding or catalysis.
• The modification site is protected by the substrate.