Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center.
Arginine 56 in the beta subunit (betaArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (-SO2H) and sulfenyl (-SOH) groups of the post-translationally modified cysteine residues in the catalytic center. BetaArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The betaR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the betaR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV-visible absorption and light-induced Fourier transform infrared difference spectra suggest that betaArg56 is involved in the positioning of the -SO2H and -SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, betaArg56 is essential for catalysis.
Piersma
S R
SR
Biochemical Systems Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan.
Nojiri
M
M
Tsujimura
M
M
Noguchi
T
T
Odaka
M
M
Yohda
M
M
Inoue
Y
Y
Endo
I
I
D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D001120:Arginine; D006836:Hydro-Lyases; C048831:nitrile hydratase