Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.
The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.
Bech
L M
LM
Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark.
Sørensen
S B
SB
Breddam
K
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
D013381:Subtilisins; D014633:Valine