Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase.
[Omega-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5', which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes.
Fukuoka
Masaki
M
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama, Japan.
Nakanishi
Yuka
Y
Hannak
Renate B
RB
Kräutler
Bernhard
B
Toraya
Tetsuo
T
D001665:Binding Sites; D002384:Catalysis; D003038:Cobamides / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry*; D007700:Kinetics; D041121:Klebsiella oxytoca / Q000201:enzymology; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry*; D011408:Propanediol Dehydratase / Q000737:chemistry*; D014805:Vitamin B 12 / Q000031:analogs & derivatives
D003038:Cobamides; D015335:Molecular Probes; C097751:cob(II)alamin; D011408:Propanediol Dehydratase; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide; D014805:Vitamin B 12