1D1T: MUTANT OF HUMAN SIGMA ALCOHOL DEHYDROGENASE WITH LEUCINE AT POSITION 141

Citation:
Abstract
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.
PDB ID: 1D1TDownload
MMDB ID: 11130
PDB Deposition Date: 1999/9/21
Updated in MMDB: 2012/11
Experimental Method:
x-ray diffraction
Resolution: 2.4  Å
Source Organism:
Similar Structures:
Biological Unit for 1D1T: tetrameric; determined by author
Molecular Components in 1D1T
Label Count Molecule
Proteins (4 molecules)
4
Alcohol Dehydrogenase Class IV Sigma Chain(Gene symbol: ADH7)
Molecule annotation
Chemicals (41 molecules)
1
19
2
13
3
5
4
4
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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