Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and...

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Pepsin is a key digestive enzyme.
• Understanding pepsin's substrate specificity is crucial for its applications.

Purpose of the Study:

• To investigate pig pepsin's cation binding specificity in secondary subsites.
• To determine the influence of basic residues on pepsin-substrate interactions.

Main Methods:

• Pepsin-catalyzed hydrolysis of synthetic peptide substrates.
• Kinetic analysis of reaction rates (kcat) and Michaelis constants (Km).
• Systematic variation of basic residue (Lysine, Arginine) positions.

Main Results:

• Basic residues significantly impacted Km (up to 5 orders of magnitude change), affecting substrate binding.
• kcat was largely unaffected by basic residues, indicating binding-step influence.
• Observed Km values below 50 nM at pH > 5.5 and kcat/Km exceeding 10(8) M-1 s-1.

Conclusions:

• Ionic interactions in pepsin's secondary binding sites primarily influence the substrate binding step.
• This contrasts with previous findings where substrate length/hydrophobicity affected catalytic rate (kcat).