Substance P hydrolysis by human serum cholinesterase

Area of Science:

• Biochemistry
• Enzymology
• Neuroscience

Background:

• Human serum cholinesterase (butyrylcholinesterase) is known for its esterase activity.
• The neuropeptide substance P plays various roles in the nervous system.
• Previous studies showed acetylcholinesterase hydrolyzes substance P at different sites.

Purpose of the Study:

• To investigate the peptidase activity of purified human serum cholinesterase (butyrylcholinesterase) towards substance P.
• To characterize the cleavage sites and kinetics of substance P hydrolysis by butyrylcholinesterase.
• To determine if substance P interacts with cholinesterase and to compare its activity with acetylcholinesterase.

Main Methods:

• Purification of human serum cholinesterase.
• High-performance liquid chromatography (HPLC) to monitor substance P digestion.
• Kinetic analysis (Km, turnover number) of hydrolysis.
• Inhibition studies using diisopropylfluorophosphate and substance P.
• Comparison of cleavage sites with acetylcholinesterase.

Main Results:

• Highly purified human serum cholinesterase demonstrated sequential peptidase activity on substance P, yielding three product peptides.
• Specific cleavage sites were identified at Arg1-Pro2 and Lys3-Pro4, with C-terminal amide removal from Met11.
• Kinetic parameters (Km = 0.3 mM, turnover = 0.6 min-1) were determined for the initial hydrolysis.
• Both peptidase and esterase activities were inhibited by diisopropylfluorophosphate.
• Substance P competitively inhibited benzoylcholine hydrolysis, confirming direct interaction with cholinesterase.

Conclusions:

• Human serum cholinesterase possesses novel peptidase and amidase activities against substance P.
• The identified cleavage pattern differs from that of acetylcholinesterase.
• The enzyme's presence in serum, brain, and muscle suggests a potential role in the physiological regulation of substance P.