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 Å 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 Ki for 4-methylpyrazole
from 350 to 10 μM, 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
σ141L mutant enzyme was determined to 2.4 Å 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.