For trypsin and chymotrypsin, the peptide-bond cleavage specificity depends primarily on what property of the substrate-binding specificity pocket?

Introduction:
Protease specificity arises from how the substrate side chain at the P1 position fits into the enzyme’s specificity pocket. This question focuses on trypsin and chymotrypsin, which share the same catalytic triad but cut different substrates because their binding pockets differ in physical and electrostatic properties.

Given Data / Assumptions:

• Trypsin, chymotrypsin, and elastase are serine proteases with identical catalytic chemistry.
• Substrate recognition differs mainly at the specificity pocket that binds the side chain N-terminal to the scissile bond (P1).
• Catalytic residues (Ser-His-Asp) are conserved and do not dictate which substrates are chosen.

Concept / Approach:
Trypsin’s pocket contains a negatively charged Asp that attracts Lys/Arg; chymotrypsin has a large hydrophobic pocket accommodating bulky aromatics; elastase has a shallow, occluded pocket favoring small residues. Thus, size, shape, and charge of the pocket determine cleavage preference, not the mere presence of Ser 195 or the water content per se.

Step-by-Step Solution:

Verification / Alternative check:
Mutational swaps of pocket residues interconvert specificities (e.g., trypsin↔chymotrypsin), demonstrating causality of pocket chemistry rather than catalytic triad identity.

Why Other Options Are Wrong:

• Ser 195 proximity: present in all serine proteases and does not confer specificity.
• Low-barrier H-bond: influences catalysis rate, not residue preference.
• Absence of water: unrealistic; water participates in deacylation.
• Covalent pre-modification: not a general requirement for these enzymes.

Common Pitfalls:
Equating catalytic efficiency with specificity or confusing P1 vs other subsites (P2, P3, etc.).

Final Answer:
Size, shape, and charge (chemical environment) of the specificity pocket