X-ray structure of human class IV σσ alcohol dehydrogenase. Structural basis for substrate specificity

Peiguang Xie, Stephen H. Parsons, David C. Speckhard, William F. Bosron, Thomas Hurley

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Abstract

The structural determinants of substrate recognition in the human class IV, or σσ, alcohol dehydrogenase (ADH) isoenzyme were examined through x- ray crystallography and site-directed mutagenesis. The crystal structure of σσ ADH complexed with NAD+ and acetate was solved to 3-Å resolution. The human β1β1 and σσ ADH isoenzymes share 69% sequence identity and exhibit dramatically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology within the σσ substrate-binding pocket, relative to the β1β1 isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the σσ structure, appears to be twisted relative to its position in the β1β1 isoenzyme. In conjunction with movements of Thr-48 and Phe-93, this twist widens the substrate pocket in the vicinity of the catalytic zinc and may contribute to this isoenzyme's high K(m) for small substrates. The presence of Met-57, Met- 141, and Phe-3O9 narrow the middle region of the σσ substrate pocket and may explain the substantially decreased K(m) values with increased chain length of substrates in σσ ADH. The kinetic properties of a mutant σσ enzyme (σ309L317A) suggest that widening the middle region of the substrate pocket increases K(m) by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.

Original languageEnglish
Pages (from-to)18558-18563
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number30
DOIs
StatePublished - 1997

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Substrate Specificity
Isoenzymes
X-Rays
Alcohol Dehydrogenase
X rays
Substrates
NAD
Hexanols
Crystallography
Niacinamide
Enzymes
Site-Directed Mutagenesis
Vitamin A
Zinc
Acetates
Mutagenesis
Kinetics
Alcohols
alcohol dehydrogenase IV
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

X-ray structure of human class IV σσ alcohol dehydrogenase. Structural basis for substrate specificity. / Xie, Peiguang; Parsons, Stephen H.; Speckhard, David C.; Bosron, William F.; Hurley, Thomas.

In: Journal of Biological Chemistry, Vol. 272, No. 30, 1997, p. 18558-18563.

Research output: Contribution to journalArticle

Xie, Peiguang ; Parsons, Stephen H. ; Speckhard, David C. ; Bosron, William F. ; Hurley, Thomas. / X-ray structure of human class IV σσ alcohol dehydrogenase. Structural basis for substrate specificity. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 30. pp. 18558-18563.
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