Methionine-141 directly influences the binding of 4-methylpyrazole in human σσ alcohol dehydrogenase

Peiguang T. Xie, Thomas D. Hurley

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Abstract

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human σσ isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human β1β1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human σσ alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human β1β1 isoenzyme structure indicated that the side-chain position for Met141 in σσ ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in σσ ADH to Leu (σ141L) lowers the K(i) for 4-methylpyrazole from 350 to 10 μM, while having a much smaller effect on the K(i) for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the σ141L mutant enzyme was determined to 2.4 A resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type σσ ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4- methylpyrazole binding to the human ADH isoenzymes.

Original languageEnglish (US)
Pages (from-to)2639-2644
Number of pages6
JournalProtein Science
Volume8
Issue number12
StatePublished - Dec 1 1999

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Keywords

  • Alcohol dehydrogenase
  • Enzyme kinetics
  • Pyrazole
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry

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