The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

Oded Kleifeld, Shu Ping Shi, Raz Zarivach, Miriam Eisenstein, Irit Sagi

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)468-479
Number of pages12
JournalProtein Science
Volume12
Issue number3
DOIs
StatePublished - 1 Mar 2003
Externally publishedYes

Keywords

  • Active site
  • Alcohol dehydrogenase
  • Glutamate
  • Metalloenzyme
  • X-ray absorption

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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