Premium
Post‐translational modification is essential for catalytic activity of nitrile hydratase
Author(s) -
Murakami Taku,
Nojiri Masaki,
Nakayama Hiroshi,
Dohmae Naoshi,
Takio Koji,
Odaka Masafumi,
Endo Isao,
Nagamune Teruyuki,
Yohda Masafumi
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.5.1024
Subject(s) - nitrile hydratase , chemistry , sulfinic acid , cysteine , sulfenic acid , nitrile , rhodococcus rhodochrous , biochemistry , rhodococcus , ferric , enzyme , organic chemistry
Nitrile hydratase from Rhodococcus sp. N‐771 is an αβ heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, αCys112 and αCys114 are modified to a cysteine sulfinic acid (Cys‐SO 2 H) and a cysteine sulfenic acid (Cys‐SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The αβ complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI‐LC/MS analysis showed that the anaerobically reconstituted αβ complex did not have the modification of αCys112‐SO 2 H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112‐SO 2 H. Furthermore, ESI‐LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that αCys114 is modified to Cys‐SOH together with the sulfinic acid modification of αCys112. These results suggest that αCys112 and αCys114 are spontaneously oxidized to Cys‐SO 2 H and Cys‐SOH, respectively, and αCys112‐SO 2 H is responsible for the catalytic activity solely or in combination with αCys114‐SOH.