A clogged gutter mechanism for protease inhibitors

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Serine proteases are crucial enzymes involved in numerous physiological processes.
• Understanding their inhibition mechanisms is vital for developing therapeutics.
• Classical peptide inhibitors offer insights into enzyme-inhibitor interactions.

Purpose of the Study:

• To elucidate the mechanism of a classical peptide inhibitor of serine proteases.
• To investigate the role of acyl-enzyme intermediates in protease inhibition.
• To determine the nucleophilic attack angle in Michaelis complexes.

Main Methods:

• Kinetic analysis of enzyme-inhibitor interactions.
• Characterization of acyl-enzyme intermediate formation and hydrolysis.
• Structural analysis of enzyme-inhibitor complexes.

Main Results:

• The inhibitor rapidly formed an acyl-enzyme intermediate, significantly slower than substrate hydrolysis.
• Tight and oriented binding of the cleaved peptide prevented acyl-enzyme hydrolysis, favoring the reverse reaction.
• This mechanism is common to tight-binding serine protease inhibitors mimicking substrates.
• The nucleophilic attack angle in Michaelis complexes was determined to be approximately 90 degrees.

Conclusions:

• The studied peptide inhibitor functions by forming a stable acyl-enzyme intermediate.
• Tight binding and product inhibition are key features of this inhibitor class.
• The determined nucleophilic attack angle provides insights into the catalytic mechanism of serine proteases.