[Conformational flexibility of enzyme active sites]

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Enzyme inactivation during denaturation often precedes global conformational changes.
• Denaturation rates indicate faster inactivation than structural alterations in the enzyme molecule.

Purpose of the Study:

• To investigate the relationship between enzyme inactivation and conformational changes during denaturation.
• To explore the role of active site flexibility in enzyme catalysis and activity.

Main Methods:

• Comparative studies of enzyme conformation and activity during denaturation.
• Utilizing fluorescent and spin probes to detect active site conformational changes in creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, and ribonuclease A.
• Analyzing ribonuclease A susceptibility to proteolysis and identifying cleavage points via peptide fragment analysis.

Main Results:

• Enzyme inactivation rates are significantly faster than global conformational changes under identical denaturation conditions.
• Direct evidence of active site conformational changes was observed using probes in multiple enzymes.
• Ribonuclease A showed increased proteolysis susceptibility in dilute GuHCl, indicating active site loosening without gross unfolding.

Conclusions:

• Enzyme active sites are proposed to be more flexible than the overall enzyme structure due to weaker binding forces.
• Active site flexibility is essential for enzyme catalysis, potentially enabling different conformational states during catalytic cycles.
• Conformational changes, particularly at the active site, accompany enzyme catalysis and activation.