Catalysis by human leukocyte elastase: proton inventory as a mechanistic probe

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein chemistry

Background:

• Human leukocyte elastase (HLE) is a serine protease implicated in various physiological and pathological processes.
• Understanding the catalytic mechanisms of HLE is crucial for developing targeted inhibitors and therapeutic strategies.
• Proton inventory studies are valuable for elucidating the transition states of enzyme-catalyzed reactions.

Purpose of the Study:

• To investigate the catalytic mechanisms of human leukocyte elastase (HLE) using proton inventory studies.
• To determine how substrate structure influences the HLE-catalyzed hydrolysis of peptide derivatives.
• To elucidate the role of the catalytic triad and proton transfer in the rate-limiting steps.

Main Methods:

• Proton inventory measurements were conducted by varying the H2O/D2O solvent composition.
• Kinetic parameters (k2/Ks) were determined for the hydrolysis of various peptide thiobenzyl esters and p-nitroanilides.
• Data were analyzed using models that account for solvent reorganization and partial rate limitation.

Main Results:

• Proton inventories for p-nitroanilides showed dome-shaped dependencies, indicating partial rate limitation by physical and chemical steps.
• Linear proton inventories for dipeptide substrates suggest simple general-base catalysis with single proton transfer.
• Bowl-shaped proton inventories for tri- and tetrapeptide substrates indicate a more complex mechanism involving the catalytic triad and two proton transfers.

Conclusions:

• The catalytic mechanism of HLE is substrate-dependent, with minimal substrates utilizing simpler pathways.
• Complex peptide substrates engage the full catalytic triad, involving multiple proton transfers in the rate-limiting transition state.
• Acyl-enzyme hydrolysis steps are not solely rate-limiting for longer peptides, with physical steps also contributing.