The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis.
Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.
Kleifeld
Oded
O
Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
Shi
Shu Ping
SP
Zarivach
Raz
R
Eisenstein
Miriam
M
Sagi
Irit
I
D015502:Absorptiometry, Photon / Q000379:methods; D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation
D018698:Glutamic Acid; D000426:Alcohol Dehydrogenase