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A naturally variable residue in the S1 subsite of M1‐family aminopeptidases modulates catalytic properties and promotes functional specialization
Author(s) -
Klemba Michael,
Ragheb Daniel,
Schubot Florian,
Dalal Seema
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.561.11
Subject(s) - residue (chemistry) , dipeptide , mutant , chemistry , stereochemistry , aminopeptidase , biochemistry , enzyme , alanine , peptide , amino acid , leucine , gene
The objective of this study was to identify principles governing the substrate specificity of M1‐family aminopeptidases at the S1 subsite, which interacts with the sidechain of the first residue of peptide substrates. By sequence comparison, we identified a residue in the S1 subsite that is highly variable and hypothesized that changes in this residue modulate S1 specificity. To test this hypothesis, we generated a large number of mutants at this position in the aminopeptidase PfA‐M1 from Plasmodium falciparum and in PepN from Escherichia coli . The kinetic properties of each mutant were characterized using a panel of natural dipeptide substrates. These studies revealed a complex interplay between the identity of the variable S1 residue and the kinetic properties of the enzyme. A dramatic shift in substrate specificity favoring the straight‐chain P1 sidechains Arg, Lys and Met could be effected by changing the S1 residue to proline. Structural analysis revealed that the proline residue induced a 1.1 Å shift of the polypeptide backbone, which resulted in constriction of the cylindrical S1 subsite. Together, the biochemical and structural data suggest that variation of a residue of the S1 subsite can modulate the catalytic properties of M1‐family aminopeptidases and ultimately contribute to functional specialization. This work was supported by National Institutes of Health grant AI077638.