A Mn(II)-Mn(II) center in human prolidase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Human prolidase catalyzes Xaa-Pro/Hyp peptide bond hydrolysis, crucial for imino acid recycling and extracellular matrix remodeling.
• The precise metal cofactor composition for maximal prolidase activity remains unidentified.
• Prolidase dysfunction is linked to carcinogenesis and genetic mutations cause connective tissue disorders.

Purpose of the Study:

• To identify the metal cofactor and its organization within human recombinant prolidase (hRecProl).
• To investigate the role of the metal cofactor in protein folding and conformational changes.
• To elucidate the structural requirements for human holo-prolidase activity.

Main Methods:

• Electron Paramagnetic Resonance (EPR) spectroscopy.
• Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
• Thermal denaturation, Circular Dichroism (CD)/fluorescence spectroscopy, and limited proteolysis.

Main Results:

• A dinuclear manganese center (Mn(II)-Mn(II)) was identified as the protein cofactor in hRecProl.
• Manganese is essential for correct protein folding.
• A conformational modification is required for the transition from apo- to Mn(II)-loaded enzyme.

Conclusions:

• The study identifies the Mn(II)-Mn(II) center as the active cofactor in human prolidase.
• The metal cofactor is critical for proper protein folding and conformational stability.
• These findings enhance understanding of human holo-prolidase structure-function relationships.