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The conserved Glu‐60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis
Author(s) -
Kleifeld Oded,
Shi Shu Ping,
Zarivach Raz,
Eisenstein Miriam,
Sagi Irit
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0221603
Subject(s) - chemistry , alcohol dehydrogenase , zinc , catalysis , stereochemistry , alanine , enzyme , residue (chemistry) , cofactor , active site , alcohol , turnover number , circular dichroism , biochemistry , organic chemistry , amino acid
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.