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Mutating the buried glutamate‐267 in horse liver alcohol dehydrogenase activates the enzyme and locks it in the open conformation
Author(s) -
Plapp Bryce V.,
Gogerty David S.,
Kim Yun Ho
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a646
Subject(s) - nad+ kinase , chemistry , cofactor , alcohol dehydrogenase , stereochemistry , enzyme , acetaldehyde , active site , oxidoreductase , conformational change , allosteric regulation , binding site , dehydrogenase , biochemistry , ethanol
Glutamate‐267 is highly conserved in alcohol dehydrogenases and is suggested to be involved in a protein promoting vibration that facilitates catalysis. The residue is buried in the coenzyme binding domain. The Glu267Asn and Glu267His substitutions increase modestly the turnover numbers with ethanol and acetaldehyde as substrates, greatly increase the dissociation constants for the coenzymes NAD + and NADH, and significantly decrease catalytic efficiencies. Large substrate isotope effects for oxidation of ethanol or benzyl alcohol suggest that hydride transfer is a major rate‐limiting step in catalysis. The enzymes are activated relative to wild‐type enzyme because coenzyme is released faster. X‐Ray crystallography shows that the structures of the mutated enzymes are similar to the open conformation of the wild‐type apo‐enzyme and that Asn‐267 or His‐267 are accommodated with local changes in the structure. Interestingly, NAD or some degradation product of NAD is found in the coenzyme binding site, but electron density is not definitive for positioning the nicotinamide ribose moiety. Furthermore, when the Glu267His enzyme is co‐crystallized with added NAD + and 2,2,2‐trifluoroethanol, the structure is also in the open conformation. The substitutions of Glu‐267 appears to prevent the conformational change to a closed form that is observed with wild‐type enzyme when it binds coenzyme. Affinity for coenzyme binding may be decreased because of small alterations in the binding pocket and impaired coupling of the binding with the conformational change. The studies support an important role for Glu‐267, but the modest changes in the rate constant for hydride transfer show that this residue is not critical for catalytic rate enhancement. (Supported by NIH grant AA00279)