TY - JOUR
T1 - Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme
T2 - Evidence that the substrate-coordinated metal ion is calcium
AU - Toraya, Tetsuo
AU - Honda, Susumu
AU - Mori, Koichi
PY - 2010/8/24
Y1 - 2010/8/24
N2 - The X-ray analyses of coenzyme B12-dependent diol dehydratase revealed two kinds of electron densities that correspond to metal ions in the active site. One is directly coordinated by substrate [Shibata, N., et al. (1999) Structure 7, 997-1008] and the other located near the adenine ring of the coenzyme adenosyl group [Masuda, J., et al. (2000) Structure 8, 775-788]. Both have been assigned as potassium ions, although the coordination distances of the former are slightly shorter than expected. We examined the possibility that the enzyme is a metalloenzyme. Apodiol dehydratase was strongly inhibited by incubation with EDTA and EGTA in the absence of substrate. The metal analysis revealed that the enzyme contains ∼2 mol of tightly bound calcium per mole of enzyme. The calcium-deprived, EDTA-free apoenzyme was obtained by the EDTA treatment, followed by ultrafiltration. The activity of the calcium-deprived apoenzyme was dependent on Ca2+ when assayed with 1 mM substrate. The Km for Ca2+ evaluated in reconstitution experiments was 0.88 μM. These results indicate that the calcium is essential for catalysis. Ca2+ showed a significant stabilizing effect on the calcium-deprived apoenzyme as well. It was thus concluded that the substrate-coordinated metal ion is not potassium but calcium. The potassium ion bound near the adenine ring would be the essential one for the diol dehydratase catalysis. Therefore, this enzyme can be considered to be a metal-activated metalloenzyme.
AB - The X-ray analyses of coenzyme B12-dependent diol dehydratase revealed two kinds of electron densities that correspond to metal ions in the active site. One is directly coordinated by substrate [Shibata, N., et al. (1999) Structure 7, 997-1008] and the other located near the adenine ring of the coenzyme adenosyl group [Masuda, J., et al. (2000) Structure 8, 775-788]. Both have been assigned as potassium ions, although the coordination distances of the former are slightly shorter than expected. We examined the possibility that the enzyme is a metalloenzyme. Apodiol dehydratase was strongly inhibited by incubation with EDTA and EGTA in the absence of substrate. The metal analysis revealed that the enzyme contains ∼2 mol of tightly bound calcium per mole of enzyme. The calcium-deprived, EDTA-free apoenzyme was obtained by the EDTA treatment, followed by ultrafiltration. The activity of the calcium-deprived apoenzyme was dependent on Ca2+ when assayed with 1 mM substrate. The Km for Ca2+ evaluated in reconstitution experiments was 0.88 μM. These results indicate that the calcium is essential for catalysis. Ca2+ showed a significant stabilizing effect on the calcium-deprived apoenzyme as well. It was thus concluded that the substrate-coordinated metal ion is not potassium but calcium. The potassium ion bound near the adenine ring would be the essential one for the diol dehydratase catalysis. Therefore, this enzyme can be considered to be a metal-activated metalloenzyme.
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U2 - 10.1021/bi100561m
DO - 10.1021/bi100561m
M3 - Article
C2 - 20712378
AN - SCOPUS:77955675459
VL - 49
SP - 7210
EP - 7217
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 33
ER -