Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Oxidoreductases like protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) are crucial for cellular redox homeostasis.
• The active sites of these enzymes contain bis(cysteinyl) motifs essential for their catalytic activity and redox potential.
• Understanding the factors governing disulfide bond formation in these active site mimics is key to elucidating enzyme mechanisms.

Purpose of the Study:

• To investigate the propensity of bis(cysteinyl)octapeptides, modeling oxidoreductase active sites, to form intramolecular disulfide rings.
• To compare the cyclic monomer formation of these peptides with the redox potentials of their corresponding native enzymes.
• To explore the influence of conformational changes, induced by trifluoroethanol and denaturing conditions, on peptide disulfide ring formation.

Main Methods:

• Oxidation experiments of bis(cysteinyl)octapeptides in aqueous buffer.
• Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy to analyze secondary structures.
• Experiments conducted under varying conditions, including trifluoroethanol addition and guanidinium hydrochloride denaturation.
• Comparison of peptide behavior with native enzyme redox potentials and structural motifs.

Main Results:

• The rank order of cyclic monomer formation in peptides was inversely related to the redox potentials (Kox) of the native enzymes.
• Induction of beta-turn structures using trifluoroethanol significantly increased cyclic monomer formation (approx. 90%) across peptides, suppressing sequence-specific differences.
• Under denaturing conditions, only the thioredoxin (trx) peptide showed statistical product distribution; others exhibited increased cyclic monomer content, potentially due to hydrophobic collapse.
• Results support the role of the thioredoxin structural motif in dictating enzyme redox properties, with active site mutations affecting redox potentials primarily through conformational constraints.

Conclusions:

• The formation of intramolecular disulfide rings in oxidoreductase active site mimics is influenced by factors beyond simple amino acid sequence.
• Conformational preferences, even when induced, can override intrinsic sequence tendencies, leading to significant increases in cyclic monomer formation.
• The characteristic thioredoxin structural motif plays a critical role in the redox properties of these enzymes, a role not fully replicated by isolated peptide fragments.
• Enzyme structure-activity relationships are complex, with three-dimensional constraints in native proteins being crucial for fine-tuning redox potentials.