Higher Metal−Ligand Coordination in the Catalytic Site of Cobalt-Substituted Thermoanaerobacter brockii Alcohol Dehydrogenase Lowers the Barrier for Enzyme Catalysis

• Published in: Biochemistry (Easton) Vol. 43; no. 22; pp. 7151 - 7161
• Main Authors: Kleifeld, Oded, Rulíšek, Lubomír, Bogin, Oren, Frenkel, Anatoly, Havlas, Zdeněk, Burstein, Yigal, Sagi, Irit
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-06-2004
• Subjects: Alcohol Dehydrogenase - chemistry; Alcohol Dehydrogenase - metabolism; Bacteria, Anaerobic - enzymology; Catalysis; Catalytic Domain; Cobalt - metabolism; Crystallography, X-Ray; Ligands; Thermoanaerobacter brockii; Water - metabolism; Zinc - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0302696

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP+/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450−458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal−ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal−ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.