PMID- 10631979
OWN - NLM
STAT- MEDLINE
DCOM- 20000214
LR  - 20181130
IS  - 0961-8368 (Print)
IS  - 0961-8368 (Linking)
VI  - 8
IP  - 12
DP  - 1999 Dec
TI  - Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma
      sigma alcohol dehydrogenase.
PG  - 2639-44
AB  - 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.
FAU - Xie, P T
AU  - Xie PT
AD  - Department of Biochemistry and Molecular Biology, Indiana University School of
      Medicine, Indianapolis 46202-5122, USA.
FAU - Hurley, T D
AU  - Hurley TD
LA  - eng
SI  - PDB/1DIS
SI  - PDB/1DIT
GR  - AA07117/AA/NIAAA NIH HHS/United States
GR  - AA07611/AA/NIAAA NIH HHS/United States
GR  - AA10399/AA/NIAAA NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Protein Sci
JT  - Protein science : a publication of the Protein Society
JID - 9211750
RN  - 0 (Isoenzymes)
RN  - 0 (Pyrazoles)
RN  - 83LCM6L2BY (Fomepizole)
RN  - AE28F7PNPL (Methionine)
RN  - EC 1.1.1.1 (Alcohol Dehydrogenase)
RN  - GMW67QNF9C (Leucine)
SB  - IM
MH  - Alcohol Dehydrogenase/*chemistry/genetics
MH  - Amino Acid Substitution
MH  - Binding Sites
MH  - Crystallography, X-Ray
MH  - Fomepizole
MH  - Humans
MH  - Isoenzymes/chemistry/genetics
MH  - Kinetics
MH  - Leucine/chemistry
MH  - Methionine/*chemistry
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Pyrazoles/*chemistry
MH  - Substrate Specificity
PMC - PMC2144219
EDAT- 2000/01/13 09:00
MHDA- 2000/02/19 09:00
CRDT- 2000/01/13 09:00
PHST- 2000/01/13 09:00 [pubmed]
PHST- 2000/02/19 09:00 [medline]
PHST- 2000/01/13 09:00 [entrez]
AID - 10.1110/ps.8.12.2639 [doi]
PST - ppublish
SO  - Protein Sci. 1999 Dec;8(12):2639-44. doi: 10.1110/ps.8.12.2639.