Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. 1999 Dec; 8(12): 2639–2644.
PMCID: PMC2144219

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


Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human sigma sigma isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human beta1beta1 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 sigma sigma alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human beta1beta1 isoenzyme structure indicated that the side-chain position for Met141 in sigma sigma ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in sigma sigma ADH to Leu (sigma141L) lowers the Ki for 4-methylpyrazole from 350 to 10 microM, while having a much smaller effect on the Ki 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 sigma141L 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 sigma sigma 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.

Full Text

The Full Text of this article is available as a PDF (708K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Al-Karadaghi S, Cedergren-Zeppezauer ES, Hövmoller S. Refined crystal structure of liver alcohol dehydrogenase-NADH complex at 1.8 A resolution. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):793–807. [PubMed]
  • Cleland WW. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. [PubMed]
  • Davis GJ, Bosron WF, Stone CL, Owusu-Dekyi K, Hurley TD. X-ray structure of human beta3beta3 alcohol dehydrogenase. The contribution of ionic interactions to coenzyme binding. J Biol Chem. 1996 Jul 19;271(29):17057–17061. [PubMed]
  • Edenberg HJ, Moss LG, Rutter WJ. Convenient vectors for cloning and sequencing EcoRI and HindIII fragments. Gene. 1987;58(2-3):297–298. [PubMed]
  • Ehrig T, Bosron WF, Li TK. Alcohol and aldehyde dehydrogenase. Alcohol Alcohol. 1990;25(2-3):105–116. [PubMed]
  • Eklund H, Nordström B, Zeppezauer E, Söderlund G, Ohlsson I, Boiwe T, Söderberg BO, Tapia O, Brändén CI, Akeson A. Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution. J Mol Biol. 1976 Mar 25;102(1):27–59. [PubMed]
  • Eklund H, Samama JP, Wallén L. Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase. Biochemistry. 1982 Sep 28;21(20):4858–4866. [PubMed]
  • Jiang JS, Brünger AT. Protein hydration observed by X-ray diffraction. Solvation properties of penicillopepsin and neuraminidase crystal structures. J Mol Biol. 1994 Oct 14;243(1):100–115. [PubMed]
  • Kedishvili NY, Bosron WF, Stone CL, Hurley TD, Peggs CF, Thomasson HR, Popov KM, Carr LG, Edenberg HJ, Li TK. Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase. Active-site amino acid sequence explains substrate specificity compared with liver isozymes. J Biol Chem. 1995 Feb 24;270(8):3625–3630. [PubMed]
  • Theorell H, Yonetani T, Sjöberg B. On the effects of some heterocyclic compounds on the enzymic activity of liver alcohol dehydrogenase. Acta Chem Scand. 1969;23(1):255–260. [PubMed]
  • Vallee BL, Bazzone TJ. Isozymes of human liver alcohol dehydrogenase. Isozymes Curr Top Biol Med Res. 1983;8:219–244. [PubMed]
  • Xie P, Parsons SH, Speckhard DC, Bosron WF, Hurley TD. X-ray structure of human class IV sigmasigma alcohol dehydrogenase. Structural basis for substrate specificity. J Biol Chem. 1997 Jul 25;272(30):18558–18563. [PubMed]
  • Yasunami M, Chen CS, Yoshida A. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7610–7614. [PMC free article] [PubMed]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...