Active site structural features for chemically modified forms of rhodanese

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Rhodanese catalyzes reactions through two main forms: sulfur-free and sulfur-substituted (persulfide-containing).
• Understanding the structural dynamics of these forms is crucial for elucidating enzyme mechanisms.

Purpose of the Study:

• To investigate the structural impact of active site modifications on sulfur-free rhodanese.
• To compare the structures of sulfur-free and sulfur-substituted rhodanese in both solution and crystalline states.

Main Methods:

• Crystallization of sulfur-free rhodanese.
• Chemical modification of sulfur-free rhodanese using hydrogen peroxide and monoiodoacetate.
• X-ray crystallography to determine enzyme structures.
• Comparison of structural data between different enzyme forms.

Main Results:

• The crystal structure of sulfur-free rhodanese closely resembles the sulfur-substituted form.
• Hydrogen peroxide inactivates sulfur-free rhodanese by oxidizing the active site's sulfhydryl group to a sulfenyl group.
• Reaction with monoiodoacetate induces a conformation in Cys-247 that mimics interactions seen in the sulfur-substituted enzyme.

Conclusions:

• Active site modifications of sulfur-free rhodanese do not induce significant global protein structural changes.
• The structural similarity between sulfur-free and sulfur-substituted rhodanese is maintained in both solution and crystal states.
• These findings support a model where rhodanese maintains a consistent overall structure throughout its catalytic cycle.