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Understanding the Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN)
Author(s) -
Crawford Patrick,
Kimberley Butnik,
Kassl Christopher,
Nelson Nicole,
Anliker Mitchell,
Golich Frank C,
Crowder Michael W
Publication year - 2012
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.26.1_supplement.963.8
Subject(s) - tyrosine , aminopeptidase , chemistry , amino acid , biochemistry , leucine
Aminopeptidase N (PepN) from E. coli is an M1 class, Zn(II)‐dependent peptidase with broad specificity. PepN was mechanistically characterized in an effort to understand the role of tyrosine 381. pH Dependence kinetic studies revealed a log k cat versus pH and k cat /K M versus pH curves with inflection points at a pH of 9.0. Solvent isotope studies suggest at least one rate‐limiting proton transfer in the mechanism. In an effort to characterize the functional roles of binding region components, several active‐site amino acids were mutated. A tyrosine to alanine mutated PepN (Y381A) was successfully over‐expressed, purified, and characterized. Y381A‐PepN maintained Zn(II) binding efficiency but exhibited greater than 900‐fold loss of activity ( k cat ) when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product. This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Miami University.