Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference

Area of Science:

• Enzymology
• Protein Engineering
• Biochemistry

Background:

• Subtilisins are serine proteases with broad substrate specificity.
• The role of residue 104 in subtilisin's P4 substrate binding has been previously hypothesized.

Purpose of the Study:

• To investigate the role of the amino acid residue at position 104 in subtilisin's P4 substrate binding.
• To determine if residue 104 solely dictates P4 specificity.

Main Methods:

• Site-directed mutagenesis was used to replace Val104 in subtilisin 309 with various amino acids (Ala, Arg, Asp, Phe, Ser, Trp, Tyr).
• Enzyme activity and substrate specificity were analyzed with different P4 residues.

Main Results:

• Subtilisin 309 exhibited a strong preference for aromatic groups at P4, irrespective of the residue at position 104.
• Hydrophilic residues at position 104 did not influence binding of hydrophobic or hydrophilic P4 residues.
• Hydrophobic residues at position 104 significantly enhanced binding of hydrophobic P4 side chains.
• An exception was observed with Asp at P4, where Arg at position 104 dramatically increased substrate preference, likely via conformational change.

Conclusions:

• The amino acid residue at position 104 is not the sole determinant of P4 specificity in subtilisins.
• Residue 104 is mobile and interacts with the P4 binding site only when favorable interactions with the substrate's P4 side chain are possible.
• Hydrophobic residues at position 104 play a significant role in binding hydrophobic P4 side chains by modulating pocket hydrophobicity or size.