Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme: evidence that the substrate-coordinated metal ion is calcium.
The X-ray analyses of coenzyme B(12)-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 approximately 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 Ca(2+) when assayed with 1 mM substrate. The K(m) for Ca(2+) evaluated in reconstitution experiments was 0.88 muM. These results indicate that the calcium is essential for catalysis. Ca(2+) 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.
Toraya
Tetsuo
T
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan. toraya@cc.okayama-u.ac.jp
Honda
Susumu
S
Mori
Koichi
K
D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004492:Edetic Acid / Q000378:metabolism; D004533:Egtazic Acid / Q000378:metabolism; D004795:Enzyme Stability; D041121:Klebsiella oxytoca / Q000201:enzymology*; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011408:Propanediol Dehydratase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000378:metabolism
D008667:Metalloproteins; D008670:Metals; D004533:Egtazic Acid; D004492:Edetic Acid; D011408:Propanediol Dehydratase; D014805:Vitamin B 12; D002118:Calcium