TY - JOUR
T1 - Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus
AU - Matsue, Tomokazu
AU - Yamada, Hiroshi
AU - Chang, Hsien Chang
AU - Uchida, Isamu
AU - Nagata, Kazuhiko
AU - Tomita, Kosuke
N1 - Funding Information:
We thank Dr. J.R. Selman, a visiting professor of our faculty, for helpful suggestions. The present study was partly supported by grant-in-Aids for Scientific Research (Nos. 0140067, 01750746 and 01850175) from the Ministry of Education, Science and Culture, Japan.
PY - 1990/3/29
Y1 - 1990/3/29
N2 - Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-l-ethanol (FEA), N,N,N′,N′-tetramethylphenylenediamine (TMPD), Co(Phen)32+ and Fe(CN)64-) catalyzed by diaphorase (NADH:acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (k0), which are difficult to determine by the conventional spectrophotometric method. Small Km) and large k0 values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)33+ and Fe(CN)63-. It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.
AB - Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-l-ethanol (FEA), N,N,N′,N′-tetramethylphenylenediamine (TMPD), Co(Phen)32+ and Fe(CN)64-) catalyzed by diaphorase (NADH:acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (k0), which are difficult to determine by the conventional spectrophotometric method. Small Km) and large k0 values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)33+ and Fe(CN)63-. It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.
KW - Cyclic voltammetry
KW - Diaphorase
KW - Digital simulation
KW - Electron mediator
KW - Electron-transfer
UR - http://www.scopus.com/inward/record.url?scp=0025213284&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025213284&partnerID=8YFLogxK
U2 - 10.1016/0167-4838(90)90006-2
DO - 10.1016/0167-4838(90)90006-2
M3 - Article
C2 - 2317516
AN - SCOPUS:0025213284
SN - 0167-4838
VL - 1038
SP - 29
EP - 38
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 1
ER -