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Post-translational modification is essential for catalytic activity of nitrile hydratase

Published online by Cambridge University Press:  01 May 2000

TAKU MURAKAMI ,
MASAKI NOJIRI ,
HIROSHI NAKAYAMA ,
MASAFUMI ODAKA ,
MASAFUMI YOHDA ,
NAOSHI DOHMAE ,
KOJI TAKIO ,
TERUYUKI NAGAMUNE  and
ISAO ENDO
TAKU MURAKAMI
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan
MASAKI NOJIRI
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
HIROSHI NAKAYAMA
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
MASAFUMI ODAKA
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
MASAFUMI YOHDA
Affiliation:
Department of Biotechnology and Life Science, Faculty of Technology, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 184-8588, Japan
NAOSHI DOHMAE
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
KOJI TAKIO
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
TERUYUKI NAGAMUNE
Affiliation:
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan
ISAO ENDO
Affiliation:
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
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Abstract

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-SO2H) 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-SO2H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112-SO2H. 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-SO2H and Cys-SOH, respectively, and αCys112-SO2H is responsible for the catalytic activity solely or in combination with αCys114-SOH.

Keywords

cysteine-sulfenic acidcysteine-sulfinic acidnitrile hydrationnonheme ironoxidationpost-translational modification
Type
Research Article
Information
Protein Science , Volume 9 , Issue 5 , May 2000 , pp. 1024 - 1030
Copyright
2000 The Protein Society

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